Objective To explore the effect of isoflurane on the oxidative stress injury induced by beta-amyloid protein (Aβ) 25-35 in PC12 cells of the rats,and to clarify the possible protective effects of trehalose on this injury.Methods The PC12 cells of rats were randomly divided into normal control group (Control group,treated with normal cell culture medium),isoflurane group (Iso group,treated with 2% isoflurane),Aβ25-35 group(Aβ group,treated with 10 μmol·L^-1 Aβ25-35),isoflurane+Aβ25-35 group (Iso+Aβ group,treated with 2% isoflurane and 10 μmol·L^-1 Aβ25-35),isoflurane+Aβ25-35+trehalose group (Iso+Aβ+Tre group,treated with 2% isoflurane,10 μmol·L^-1 Aβ25-35 and 200 mmol·L^-1 trehalose),and trehalose group (Tre group,treated with 200 mmol·L^-1 trehalose).The survival rates of the PC12 cells were detected by MTT assay;the apoptotic rates of the PC12 cells in various groups were determined by Hoechst 33342 staining;the levels of intracellular reactive oxygen species (ROS) of the PC12 cells were measured by DCFH-DA fluorescence assay;the levels of malondialdehyd (MDA) and the activities of superoxide dismutase (SOD),glutathion peroxidase (GSH-Px), and catalase (CAT) of the PC12 cells were detected by commercial kits.Results Compared with control group,the apoptotic rates of the cells were increased and the survival rates were decreased,and the levels of ROS and MDA and the activities of SOD,GSH-Px, and CAT in the PC12 cells were significantly decreased (P<0.05 or P<0.01) in Aβ group,Iso group and Iso+Aβ group.Compared with Iso group or Aβ group,the apoptotic rates were increased,the survival rates were decreased,the levels of ROS and MDA were increased,and the activities of SOD,GSH-Px,and CAT in the PC12 cells in Iso+Aβ group were decreased(P<0.05).Compared with Iso+Aβ group,the survival rates were increased and the apoptotic rates were decreased and the levels of ROS and MDA were decreased,and the activities of SOD,GSH-Px,and CAT in the PC12 cells in Iso+Aβ+Tre group were increased (P<0.05).Conclusion Isoflurane could aggravate the oxidative stress injury and the apoptosis induced by Aβ25-35 in the PC12 cells of the rats,and trehalose could alleviate the cytotoxicity of isoflurane via inhibition of oxidation and apoptosis.

Objective To explore the kidney damage in the rats caused by different concentrations of CdCl₂ and the role of autophagy in the kidney damage,and to clarify the mechanism of the kindney damage caused by CdCl₂.Methods 40 Wistar rats were randomly divided into blank control group,low dose group (0.2 mg·kg^-1 CdCl₂),middle dose group(0.4 mg·kg^-1 CdCl₂) and high dose group (0.8 mg·kg^-1 CdCl₂),and there were 10 rats in each group. The cadmium chloride solution was administrated by intraperitoneal injection to prepare the rat models of kidney damage. The same volume of normal saline was injected into the rats in blank control goup. The general status and body weights of all rats were observed. The kidney tissue and blood of the rats were obtained 5 weeks later. The renal function indexes including creatinine,blood urea nitrogen,and β2-microglobulin were detected;the levels of cadmium in the kidney tissue of the rats in various groups were detected by atomic absorption spectrometry;the relative expression level of LC3B and expression level of Beclin1 protein were detected by Western blotting and immunohistochemical methods,respectively.Results Obvious depression was seen in the rats in CdCl₂ groups,and the body weights of the rats in high dose group were significantly lower than those in blank control group from 3 weeks after exposure to cadmium (P<0.05);the body weights of the rats in middle dose group and high dose group were significantly lower than those in blank control group at the end of this experiment (P<0.05). The results of blood biochemical detection showed that the levels of creatinine,blood urea nitrogen, and β2-microglobulin of the rats in middle dose group and high dose group were significantly higher than those in blank control group (P<0.05),and the levels of creatinine of the rats in high dose group were significantly higher than those in low dose group (P<0.05);the levels of blood urea nitrogen of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05). The results of atomic absorption spectrometry showed that there were a small amount of cadmium accumulation of the rats in blank control group,but a large amount of metal cadmium accumulated in the kidney tissue were seen in low,middle and high dose groups and the levels of cadmium were increased with the increasing of the exposure dose;the levels of cadmium of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05);the levels of cadmium of the rats in high dose group were significantly higher than those in middle dose group (P<0.05).The immunohistochemistry results showed that the expression levels of Beclin1 protein in the kidney tissue of the rats in CdCl₂ groups were significantly higher than those in blank control group(P<0.05),and the expression levels of Beclin1 protein of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05);the expression levels of Beclin1 protein of the rats in high dose group were significantly higher than those in middle dose group (P<0.05).The Western blotting results showed that the relative expression levels of LC3B protein in the kidney tissue of the rats in CdCl₂ groups were significantly higher than those in blank control group (P<0.05),and the relative expression levels of LC3B protein in the kidney tissue of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05).Conclusion At the range from 0.2 to 0.8 mg·kg^-1,the kidney damage gets more and more serous with the increasing of the exposure dose of CdCl₂,and autophagy may be one of the mechanisms.

Objective To investigate the effects of bone marrow-derived endothelial progenitor cells(EPCs) conditioned medium(EPCs-CM) on the proliferation and osteogenic differentiation of mesenchymal stem cells(MSCs),and to clarify the mechanisms.Methods The EPCs and MSCs were isolated from bone marrow of the mice using differential adhesion method.The surface markers of EPCs and MSCs were identified by flow cytometry (FCM).The osteogenic,chondrogenic,and adipogenic induction differentiation abilities of the MSCs were identified.The function of EPCs was identified by tube formation experiment.The MSCs were divided into 0% EPCs-CM group(control group,cultured with LG-DMEM),50% EPCs-CM group(cultured with 50% EPGs-CM and 50% LG-DMEM),and 100% EPCs-CM group(cultured with 100% EPCs-CM).The proliferation activities of the MSCs in various groups were detected by MTT method;the osteogenic differentiation abilities of the MSCs in various groups were detected by Alizarin red staining.Results The FCM results showed that the third passage MSCs were strongly positive for Sca-1,CD29 and negative for CD45,CD11b and could be induced to complete differentiation process into osteoblasts and adipocytes and chondrocytes.The third passage EPCs cultured on Matrigel showed tube-like structures and highly expressed CD34,CD133 and VEGFR2.Compared with control group,the proliferation activities of the MSCs in 50% EPCs-CM group and 100% EPCs-CM group were increased(P<0.05),which presented a dose-dependent manner.The Alizarin red staining results showed the number of mineralized nodules and calcium deposition of the MSCs in 50% EPCs-CM group and 100% EPCs-CM group were higher than those in control group after cultured for 21 d(P<0.05).Conclusion The method of differential adhesion can simultaneously isolate the MSCs and EPCs.EPCs-CM can promote the proliferation and osteogenic differentiation of the MSCs.

Objective To investigate the protective effects of curcumin on the hepatic injury of the rats with experimental hepatic fibrosis,and to clarify its mechanism.Methods The hepatic fibrosis models of rats were established by bile duct ligation.21 rats were randomly divided into sham operation group(given saline),model group (given saline for model rats),curcumin group(given 400 mg·kg^-1 curcumin for model rats);there were 7 rats in each group.The activities of aspartate aminotransferase (AST),alanine aminotransferase (ALT) and the level of total bilirubin (Tbil) in serum of the rats in various groups were detected after treated for 2 weeks;the pathomorphology of liver tissue was observed by hematoxylin and eosin (HE) staining;the deposition of collagen in the liver tissue was observed by Massion staining;the expression levels of transforming growth factor-beta1 (Tgf-β1) and α-smooth muscle actin (α-Sma) proteins were detected by Western blotting method.Results Compared with sham operation group,the activities of AST,ALT and the levels of Tbil in serum of the rats in model group and curcumin group were increased(P<0.05).Compared with model group,the activities of AST,ALT and the level of Tbil were increased in curcumin group were decreased (P<0.05).The liver tissue of the rats in model group was obviously damaged with inflammatory cell infiltration,collagen deposition and bile duct proliferation;the pathological changes of liver tissue were significantly improved after curcumin treatment.The Western blotting results showed that the levels of Tgf-β1 and α-Sma proteins in the liver tissue of the rats in model group were increased compared with sham opration group(P<0.05);compared with model group,the levels of Tgf-β1 and α-Sma proteins in the liver tissue of the rats in curcumin group were decreased(P<0.05).Conclusion Curcumin shows the therapeutical effects on hepatic fibrosis,which is expected to be the drug to prevent and cure cholestatic hepatic fibrosis.

Objective To investigate the expressions of the key molecules β-catenin,c-myc,and cyclinD₁ of the Wnt/β-catenin signal pathway in the NK/T-cell lymphoma (NKTCL) tissue, and to clarify their relationships with the clinicopathological features of the NKTCL patients.Methods Real-time PCR method was used to detect the expression levels of β-catenin,c-myc,and cyclinD₁ mRNA in human NKTCL cell lines (SNK-6 and YTS) and normal NK cells;the positive expression rates of β-catenin and c-myc proteins in the NKTCL tissue and reactive hyperplasia of lymph node tissue were detected by immunohistochemistry SP method;the correlations among the expressions of β-catenin and c-myc proteins and the clinicopathological features of the NKTCL patients were analyzed;correlation analysis was used to analyze the relationship between the expressions of β-catenin and c-myc proteins of the NKTCL patients;Kaplan-Meier method was used to analyze the relationship between the expression of β-catenin and overall survival rate of the NKTCL patients.Results The positive expression rates of β-catenin,c-myc,and cyclinD₁ mRNA in the SNK-6 cell line and YTS cell line were significantly higher than those in the normal NK cells(P<0.05);the expression β-catenin and c-myc proteins in NKTCL tissue(24% and 56%) were higher than those in reactive hyperplasia of lymph node tissue (0 and 25%)(P<0.05);the expression of β-catenin was correlated with Ann Arbor stage(P<0.05),and the expression of c-myc was correlated with Ann Arbor stage and Ki-67(P<0.05);there were positive correlations between the expression of β-catenin and the expression of c-myc in the NKTCL patients(r=0.770,P<0.05);the patients with β-catenin positive expression had a shorter overall survival time than the patients with negative expression (P<0.05).Conclusion The key molecules β-catenin,c-myc,and cyclinD₁ of the Wnt/β-catenin signal pathway are highly expressed in the human NKTCL tissue,and they show positive correlations with Ann Arbor stage of the NKTCL patients.

Objective To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis and the expressions of membrane-type 1 matrixmetallo-proteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2(TIMP-2) in the myocardium tissue of the rats with diabetes mellitus(DM),and to clarify the effect of H₂S on the development of the myocardial fibrosis and its mechanism.Methods 52 rats were randomly divided into control group,DM group,DM+H₂S group and H₂S group (n=13).The rat models were induced by intraperitoneal injection of streptozotocin (STZ). The rats in control group(n=12) and DM group(n=9) were injected with saline after modeling and the rats in DM+H₂S group(n=9) and H₂S group(n=10) were administrated with 100 mg·kg^-1 NaHS.After 8 weeks,the pathomorphology of myocardium tissue of the rats in various groups were analyzed by VG staining;the expression levels of type Ⅲ collagen,MT1-MMP and TIMP-2 proteins in the myocardium tissue of the rats were analyzed by Western blotting method.Results Compared with control group,disorgnised array in the myocardial cells,collagen deposition and myocardial fibrosis of the rats were found in DM group;the expression levels of type Ⅲ collagen,MT1-MMP and TIMP-2 proteins in myocardium tissue of the rats were significantly increased (P<0.05).Compared with DM group,the myocardial fibrosis was improved of the rats in DM+H₂S group,and the expression levels of type Ⅲ collagen,MT1-MMP and TIMP-2 proteins in the myocardium tissue of the rats in DM+H₂S group were significantly decreased (P<0.05).Conclusion H₂S could improve the myocardial fibrosis and decrease the expression of type Ⅲ collage protein in diabetic rats and its mechanism may be related to the down-regulation of MT1-MMP and the improvement of the imbalance of MT1-MMP/TIMP-2.

Objective To discuss the relationship between miR-18a and the irradiation sensitivity of the colorectal cancer SW116 cells,and to elucidate the possible mechanism of the effects of miR-18a on the apoptosis and autophagy of the cells.Methods The SW116 cells were divided into miR-18a NC group,miR-18a mimicgroup, miR-18a NC+4Gy group,and miR-18a mimic+4Gy group. The expression of miR-18a in SW116 cells was detected by qRT-PCR before and after irradition;colony-forming assay was used to detect the effect of miR-18a on the irradiation sensitivity of the SW116 cells;GFPLC3 morphological assay was used to detect the autophagy rate of SW116 cells;the apoptotic rate of SW116 cells was analyzed by flow cytometry;the target gene of miR-18a was predicted by bioinformatics methods and dual-luciferase reporter assay was used to demonstrate the binding of miR-18a and 3'UTR targets;the expression of ataxia-telangiectasia mutated(ATM) protein in SW116 cells was measured by Western blotting method.Results compared with before irradation,the expression level of miR-18a in the SW116 cells after irradation was decreased obviously (P<0.05). The colony-forming assay results showed that the irradiation sensitivity of the SW116 cells in miR-18a mimic group was increased compared with miR-18a NC group(P<0.05),and the apoptotic rate and autophagy rate of the SW116 cells in miR-18a mimic group were increased (P<0.05). Compared with miR-18a NC+4Gy group,the expression level of ATM protein in miR-18a mimic+4Gy group was decreased.Conclusion ATM is the target gene of miR-18a in the SW116 cells. miR-18a mimic can increase the apoptosis and autophagy induced by irradition,and can increase the radiosensitivity of colorectal cancer SW116 cells.

Objective To observe the expression level of the phosphorylated microtubule-associated protein Tau (p-Tau) in the hippocampus tissue of the rats with depression,and to explore the relationship between p-Tau and depression.Methods 40 rats were randomly divided into control group (n=20) and model group (n=20).The depression models of rats were established by chronic unpredictable mild stress (CUMS),and the rats in control group were caught once a day.The behavior examination was carried out in control group and model group by sucrose preference test,open field test,and Morris water maze.The morphology of hippocampal neurons of the rats was observed by Nissl staining.At the 1st,7th,14th,and 28th day after CUMS,the rats in control group and model group were executed in batches and the expression levels of p-Tau protein in the hippocampus tissue of the rats were detected by Western blotting method.Results The sucrose preference test results showed that the sucrose consumption and the percentage of the sucrose preference of the rats in model group were lower than those in control group (P<0.01);the open field test results showed the overall walking distance,the central activity time,the up-right times,and the grooming behavior of the rats in model group were lower than those in control group (P<0.01);the Morris water maze results showed the average escape latent period of the rats in model group was higher than that in control group (P<0.01).The expression level of p-Tau protein in the hippocampus tissue of the rats in model group was lower than that in control group 1 d after CUMS(P<0.01),and was higher than that in control group after 7 d(P<0.05);the expression level of p-Tau protein in the hippocampus tissue of the rats in model group was the highest 14 d after CUMS(P<0.01).Conclusion The expression level of p-Tau protein in the hippocampus tissue of the rats with depression is decreased firstly and then increased,which may affect the stability of microtubule and destroy the steady state of cytoskeleton,thus leading to the structural changes of the hippocampal neurons.

Objective To observe the effects of nano-SiO₂ on apoptosis and cell cycle of human neuroblastoma SH-SY5Y cells,and to explore the mechanism.Methods The SH-SY5Y cells were treated with different concentrations of 90 nm SiO₂ and divided into 0,3.125,6.250,12.500,25.000, and 50.000 mg·L^-1 SiO₂ groups. The survival rates of the cells in various groups were assessed by MTT method;the apoptotic rates of SH-SY5Y cells were determined by AO/EB staining and Annexin Ⅴ-FITC/P method; the percentage of cell cycle and the level of intracellular reactive oxygen species(ROS) were tested by flow cytometry(FCM) method.Results The MTT results showed the survival rates of the cells in 6.250,12.500,25.000 and 50.000 mg·L^-1 SiO₂ groups were significantly lower than that in control group (P<0.05);the survival rates of the cells in 12.500,25.000, and 50.000 mg·L^-1 SiO₂ groups were significantly lower than that in 3.125 mg·L^-1 SiO₂ group (P<0.05); the AO/EB staining and Annexin Ⅴ-FITC/PI results showed the apoptotic rates of the cells in 6.250,12.500,25.000,and 50.000 mg·L^-1 SiO₂ groups were higher than that in control group(P<0.05);the apoptotic rates of the cells in 12.500,25.000,and 50.000 mg·L^-1 SiO₂ groups were higher than that in 3.125 mg·L^-1 group(P<0.05).The FCM results showed the levels of ROS of the cells in 3.125,6.250,12.500,25.000, and 50.000 mg·L^-1 SiO₂ groups were significantly higher than that in control group (P<0.05); compared with control group,the percentages of the cells at G₀/G₁ phase in 25.000 and 50.000 mg·L^-1 SiO₂ groups were increased (P<0.05);the percentage of the cells at G₂/M phase in 50.000 mg·L^-1 SiO₂ group was increasd (P<0.05).Conclusion Nano-SiO₂ can decrease the activity of SH-SY5Y cells and increase the apoptosis and the level of ROS,and nano-SiO₂ can interfere the cell cycle and inhibit the proliferation of SH-SY5Y cells.

Objective To observe the effect of Rhizoma atractylodis extract on the gastrointestinal motility and immune function of the rats with experimental spleen deficiency syndrome,and to elucidate the mechanism of Rhizome atractylodis extract on regulating the gastrointestinal dynamic and immune function.Methods 60 male SD rats were randomly divided into normal group,model group,low,medium and high doses of Rhizome atractylodis extract groups,domperidone group;there were 10 rats in each group.Except for normal group,the rats in other groups were fed with Xiaochengqi Decoction plus hunger to establish the spleen deficiency syndrome models.After the model establishing,the rats in low,medium and high doses of Rhizome atractylodis extract groups were gavaged with 0.5,1.0,2.0 g·mL^-1 Rhizome atraetylodis extract,respectively; the rats in domperidone group were gavaged with the domperidone suspension solution, and the rats in normal group and model group were gavaged with physiological saline.Charcoal gavage method was used to determine the ratio of stomach retention and the rate of small intestinal propulsion of the rats;intestinal perfusion method was used to detect the levels of IgA in intestinal perfusion liquid of the rats; the method of drawing blood from abdominal aorta was used to detect the levels of IgG in serum and motilin(MTL),substance P(SP),somatostatin(SS) in plasma of the rats;immunohistochemistry method was used to detect the expression levels of Toll-like receptor 4(TLR4) in duodenum,jejunum and ileum tissues.Results Compared with model group,the levels of IgA in the intestinal perfusion fluid and IgG in the serum of the rats in low,medium and high doses of Rhizome atractylodis extract groups and domperidone group were increased;the rates of stomach retention of the rats were decreased obviously,and the ratios of small intestinal propulsion of the rats were increased significantly,while the levels of MTL,SP, and SS in the plasma of the rats were increased (P<0.05 or P<0.01). Compared with low dose of Rhizome atractylodis extract group,the levels of IgA in the intestinal perfusion liquid and IgG in the serum of the rats in medium and high doses of Rhizome atractylodis extract groups were increased;the rates of stomach retention of the rats were decreased and the ratios of small intestinal propulsion of the rats were increased,while the levels of MTL,SP,and SS in the plasma of the rats were increased,and the levels of TLR4 in the duodenum,jejunum and ileum tissues were increased (P<0.05,P<0.01).Conclusion Rhizome atractylodis extract can improve the gastrointestinal motility and the immune function of the rats with experimental spleen deficiency syndrome,and has better regulation and treatment effects on the functional gastrointestinal disorders and low state of systemic and local immune function.

Objective To detect the clustered regularly interspaced short palindromic repeats (CRISPR) of Shigella,and to analyze the characteristics and homology of their repeats and spacers with complete genome Shigella in GenBank.Methods The CRISPR were obtained by PCR amplification method,and the homology of repeats and spacers was analyzed by bioinformatics;multiple sequence alignment was used to analyze the features of spacers and the relationship between the spacers and flanking sequences;BLAST was used to analyze the homology between the spacers and plasmids and phages;weblogo was used to analyze the frequency of the bases of the repeats and RNAfold was used to predict the secondary structures of the repeats RNA;homology clustering analysis of repeats was performed and BLAST was used to analyze the homology of the repeats with other bacteria.Results All 3 studied clinical strains and 9 complete genome Shigella contained different number of CRISPR.In the same CRISPR,the spacers were similar or different;the partial sequences of the spacers in some CRISPR and flanking sequences of CRISPR had the same sequences.Some spacers possible came from the splicing and gene recombination between the informational genes and operational genes.There may be relationship between the repeats which were not completely conserved and bacterial evolution.The differences of the bases of the repeats affected the length of stem,thus affected the stability of the secondary structure and the function of CRISPR.The repeats had high similarity with the several individual species of distant relationships.Conclusion The different CRISPR structures exist in Shigella,and the conservation of the repeats in different CRISPR is different.The partial sequences of some spacers have homology with the flanking sequences of CRISPR,and some spacers come from the gene splicing.

Objective To construct the E.coli expressiom system of the HBc-IsdB_50-285 fusion protein, and to explore the expression efficacy of the HBc-IsdB_50-285 fusion protein in prokaryotic system.Methods HBc-IsdB_50-285 fusion gene fragment was designed and cloned,and the expression vector pET-28-HBc-IsdB_50-285 was construct and transformed into E.coli BL21,the expression of protein was inducted by IPTG. The solubility of protein and its relative molecular mass were analyzed by SDS-PAGE method; BCA assay was used to detect the level of protein; the immune activity of the recombinant protein was detected by immune inoculation method.40 mice were divided into rIsdB+adjuvant group(n=10), HBc-IsdB_50-285+adjuvant group(n=10), HBc-IsdB_50-285 group(n=10), and PBS group(n=10).The values of special IgG of the mice were tested by ELISA method. The mice were immunized and attacked by Stathylococcus aureus Newman strain,and the immune protection rate of the recombinant protein was detected.Results The expression vector pET-28-HBc-IsdB_50-285 was successfully constructed through identification of sequencing and restriction enzyme digestion. The SDS-PAGE results showed that the recombinant protein existed in a soluble form in the supernatant with relative molecular mass about 44 000.The level of purificated protein was 1.0 g·L^-1.The ELISA results showed that the value of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group when the rIsdB were regarded as coating antigen(P<0.01);the values of antibody titer of the mice in HBc-IsdB_50-285+adjuvant group was higer than those in rIsdB+adjuvant group and HBc-IsdB_50-285 group when the HBc-IsdB_50-285 were regarded as coating antigen(P<0.05);the attack test results showed that the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group was lower than that in rIsdB+adjuvant group (P>0.05). Compared with HBc-IsdB_50-285 group, the protection rate of Staphylococcus aureus of the mice in HBC-IsdB_50-285+adjuvant group had no significant difference (P>0.05). Compared with PBS group, the protection rates of Staphylococcus aureus of the mice in HBc-IsdB50-285+adjuvant group(P<0.01) and HBc-IsdB_50-285 group(P<0.05) were increased.Conclusion A prokaryotic expression vector of HBC-IsdB_50-285 is constructed, and the HBC-IsdB_50-285 fusion protein with better biological activities is expressed in E.coli BL21 successfully.

Objective To construct three strains of anti-phage Salmonella Paratyphi A mutants by homologous recombination and knocking out genes in wild Salmonella Paratyphi A, and to analyze their biological characteristics.Methods The No.2,3,5 homology arm fragments obtained by PCR and fusion PCR were connected with pYG4 vector after digestion to construct the pYG4-2,pYG4-3,and pYG4-5 homologous recombination plasmids. Three strains of anti-phage mutants (No.2,3,5) were obtained after the plasmids were identificated by PCR sequencing and recombinated with wild Salmonella Paratyphi A. After the three mutant strains and wild Salmonella Paratyphi A were treated with different kinds and concentrations of antibiotics,different pH values and temperature and different concentrations of salt ions,respectively;the A₅₉₅ values were measured,and their sensitivities to the antibiotics,temperature,pH values and salt ions and the growth curves were analyzed.Results The results of PCR and fusion PCR amplification suggested that three homology arms were obtained. The PCR sequencing showed that the homologous recombinant plasmids pYG4-2,pYG4-3,and pYG4-5 were constructed successfully.The homologous recombination and anti-phage verification implied that three strains of anti-phage mutants were successfully gained. Three strains of anti-phage mutants gained kanamycin resistance and showed highly sensitivity to streptomycin;the No.2 mutant strain showed ampicillin resistance and the No.5 mutant strain performanced a high temperature tolerance. In addition,the growth curves of three mutants became obviously placid than that of wild Salmonella Paratyphi A,especially in the No.2 mutant.Conclusion The anti-phage mutants are successfully constructed and get some specially biological chatacteristics.

Objective To investigate the enhancement effect of resveratrol on the sensitivity of laryngeal carcinoma Hep-2 cells to cisplatin(DDP) and its mechanism.Methods The Hep-2 cells were randomly divided into blank control group and different doses(3,6,12, and 24 μmol·L^-1)of DDP groups and the cells were treated for 6,12,24,and 48 h.The suitable concentration of DDP(6 μmol·L^-1) and culture time(24 h) were chosen.The Hep-2 cells treated under the suitable condition were randomly divided into control group,DDP group,resveratrol group,sirtinol group,resveratrol+sirtinol group,resveratrol+DDP group,sirtinol+DDP group and resveratrol+sirtinol+DDP group. The survival rates of the Hep-2 cells in various groups were detected by CCK-8 assay;the expression levels of Sirt1 protein in the Hep-2 cells in various groups were determined with immunofluorescence method;the apoptotic rates of the Hep-2 cells in various groups were detected by Annexin Ⅴ-FITC Kit.Results The CCK-8 assay results showed that the survival rates of the Hep-2 cells in resveratrol group and DDP group were decreased significantly compared with control group(P<0.01);compared with DDP group,the survival rates of the Hep-2 cells in resveratrol+DDP group were decreased(P<0.01);compared with resveratrol+DDP+sirtinol group,the survival rates of the Hep-2 cells in resveratrol+DDP group were decreased(P<0.05).The immunofluorescence results showed that the mean fluorescence intensity of Sirt1 protein of the cells in resveratrol+DDP group were increased significantly compared with DDP group(P<0.01);compared with resveratrol+sirtinol+DDP group,the mean fluorescence intensity of Sirt1 protein of the cells in resveratrol+DDP group was increased significantly (P<0.01).The Annexin Ⅴ-FITC Kit results showed the apoptotic rate of the cells in resveratrol+DDP group was higher than those in DDP group and resveratrol+sirtinol+DDP group(P<0.01).Conclusion Resveratrol can improve the sensitivity of Hep-2 cells to cisplatin,and its mechanism may be related to increasing the expression of Sirt1 protein.

Objective To investigate the effect of Alkaline S.chinenis polysaccharides(ASPS) on the proliferation and invasion abilities of the human colon cancer HT-29 cells,and to clarify the anti-tumor effect of ASPS.Methods 40 Wistar rates were randomly divided into control group(n=10,given saline),and 100,200,400 mg·kg^-1 ASPS groups(n=10),respectively.The serum of the rats in various groups were gotten.The HT-29 cells were treated with the serum of the rats in various groups. The inhibitory rates of proliferation and invasion abilities were tested by MTT and Transwell assay;the expression levels of bcl-2,bax,and Caspase-3 proteins in the supernatant of the cells of the rats in various groups were determined by Western blotting method.Results Compared with control group,the inhibitory rates of proliferation and invasion abilities of the HT-29 cells in 200 and 400 mg·kg^-1 ASPS groups were increased(P<0.05, P<0.01).The expression level of bcl-2 protein in 400 mg·kg^-1 ASPS group was lower than those in other groups (P<0.05),while the expression levels of bax and Caspase-3 proteins were higher than those in control group(P<0.05, P<0.01).Conclusion ASPS can inhibit the proliferation and invasion abilities of the human colon cancer HT-29 cells,and its mechanism may be related to down-regulating the expression of bcl-2 protein and up-regulating the expressions of bax and Caspase-3 proteins,thus activating the apoptotic pathway of HT-29 cells.

Objective To investigate the protective effect of Danshenlinzhi(DSLZ) on the cerebral ischemia reperfusion injury in the rats, and to explore its mechanism.Methods 60 Wistar rats were randomly divided into sham group,model group,TanⅡA group, and low,middle,high, doses (5.0,10.0,20.0 mg·kg^-1) of DSLZ groups(n=10). The thread embolism was performed to establish the rat models of focal cerebral ischemia reperfusion injury. The neurologic scores of the rats after 2 h ischemia and 22 h reperfusion were measured and the percentages of the cerebral infarction area of the rats were detected by TTC staining method; the pathomorphology of the brain tissue of the rats was detected by HE staining. The activities of superoxide dismutase (SOD) and levels of malondialdehyde(MDA),nitric oxide (NO) in the brain tissue of the rats were detected.Results Compared with sham group,the neurologic score of the rats in model group was increased(P<0.05),the percentages of the cerebral infarction area was increased(P<0.05),the activity of SOD was decreased(P<0.01) and the levels of MDA and NO were increased (P<0.01). Compared with model group,the neurologic scores of the rats in middle and high doses of DSLZ groups were decreased and the percentages of the cerebral infarction area were decreased,the activities of SOD were increased and the levels of MDA and NO were decreased(P<0.05 or P<0.01).The HE staining results showed the membrane of neurocyte was unclear,and the cells became swelling,deformation or necrosis,severe edema in interstitium and the cell space was enlarged in model group;the nucleolus could be found clearly and there was slight edema in interstitium of the cells of the rats in high dose of DSLZ group.Conclusion DSLZ has a protective effect on the cerebral ischemia reperfusion injury of the rats,and its mechanism may be related to the anti-radical damage and lessening the neurotoxicity of NO.

Objective To explore the effect of dibutyl phthalate(DBP) on the expression of Cx43 protein and testosterone excretion in the Leydig cells of testicle in the adult rats,and to disscuss whether there is relationship between the regulation of testosterone by DBP and Cx43.Methods After the adult SD rats were killed, the cultured primary pure Leydig cells were divided into solvent control group(0.1%DMSO),DBP group (50 mg·L^-1 DBP),Cx43 enhancer group(50 mg·L^-1 DBP+10 μmol·L^-1 PGE2),and specific testosterone blocker group (40 mg·L^-1 flutamid).After cultured for 24 h,radioimmunoassay was used to measure the levels of testosterone in the Leyding cells; cell immunofluorescence and Western blotting method were performed to detect the expressions of Cx43 protein in the Leydig cells.Results Compared with DMSO group,the level of testosterone and the expression level of Cx43 protein in the Leydig cells in DBP group were decreased after 24 h exposure(P<0.05).Compared with DBP group,the testosterone level in DBP+PGE2 group had no obvious change(P>0.05),but the expression level of Cx43 protein was increased(P<0.05).Compared with DMSO group,the level of testosterone and the expression level of Cx43 protein in flutamide group were decreased significantly(P<0.05).Conclusion DBP has the effects on the Cx43 expression and testosterone excreation,and Cx43 has no inhibitory effect on the synthesis of testosterone.However,testosterone can regulate the expression of Cx43 reversely.Therefore,it's not necessary to take Cx43 as the target of DBP in adjusting the synthesis of testosterone in Leydig cells.

Objective To disscuss the effect of the acute epileptic seizure induced by pentetrazol (PTZ) on the expressions of F-actin,Calponin3,and ROCK2 in the cerebral neurons of the rats,and to illuminate the possible mechanism of preventing the F-actin from abnormal depolymerization and triggering the rearrangement of F-actin.Methods 56 immature rats aged 3 weeks were divided into control (n=6) and epilepsy (n=50) groups. The rats in control group were injected with physiological saline introperitoneally.The rats in epilepsy group were introperitoneally injected with 60 mg·kg^-1 PTZ to establish the acute epileptic seizure models.And 6 rats from successful acute epileptic seizure models were sacrificed at different time points (1,2,3,and 7 d) after modeling.The fluorescence intensity of F-actin in the internal molecular layer of hippocampus of the rats was observed by phalloidine staining labeled by Alex-488; the distributions of Calponin3 and ROCK2 in the cerebral neurons in the pallium and hippocampus of the rats were detected by immunofluorescence method; the relative expression levels of Calponin3,ROCK2,and phosphorylated ROCK2 were analyzed by Western blotting method.Results Compared with control group,the fluorescence intensity of F-actin in the hippocampal internal molecular layer of the rats in epilepsy group was decreased (P<0.05),and the dot-shaped aggregation of F-actin was disappeared 1 d after modeling.The immunofluorescence results showed that Calponin3 dispersed in the cytoplasm of the neurons of the rats in control group;however,it aggregated in the cell cortex of the neurons 7 d after modeling in epilepsy group.ROCK2 was located in a small amount of neuritis of the rats in control group,whereas a great quantity of ROCK2 was found in both of the cell body and neuritis in epilepsy group 7 d after modeling.The Western blotting results showed that the relative expression levels of Calponin3 protein in the epilepsy group were markedly decreased at different time points after modeling compared with control group (P<0.05);however,it was increased gradually with the prolongation of time and closed to the level in control group within 1 week.The relative expression levels of ROCK2 protein in the hippocampus of the rats in epilepsy group began to increase steadily from 3 to 7 d after modeling compared with control group (P<0.05);however,the relative expression levels of phosphorylated ROCK2 protein in epilepsy group were increased sustainably from 1 to 7 d after modeling compared with control group (P<0.05),and were decreased gradually in a time-dependent manner.Conclusion The acute epileptic seizure of the immature rats induced by PTZ not only leads to the abnormal depolymerization of F-actin,but also actives the RhoA/ROCK2 signal pathway simultaneously and up-regulates the expression levels of ROCK2 and Calponin3 proteins.

Objective To construct the interference vector stably silencing Rhoc gene in the ovarian cancer SKOV3 cells induced by lentivirus,and to observe its inbibitory effect on the expression of RhoC gene in the ovary cancer SKOV3 cells.Methods The ovary cancer SKOV3 cells were divided into psiHIV-RhoC1 group(transfected with Lenti-shRhoC1),psiHIV-RhoC2 group(transfected with Lenti-shRhoC2),negative control group(transfected with Lenti-NC) and blank control group(with no transfection).The expressions of green fluorescence protein in the SKOV3 cells in various groups were detected;the expression levels of RhoC mRNA in the SKOV3 cells in various groups were detected by RT-PCR method.Results There were the expressions of the green fluorescence protein in the SKOV3 cells in psiHIV-RhoC1,psiHIV-RhoC2,and negative control groups. The specific interference vector was constructed and transfected into SKOV3 cells successfully.Compared with bank control group,the expression levels of RhoC mRNA in the SKOV3 cells in psiHIV-RhoC1,psiHIV-RhoC2,and negative control groups were decreased.Conclusion The recombinant lentivirus RhoC shRNA interference vector can suppress the expression of RhoC mRNA in SKOV3 cells effectively.

Objective To investigate the effect of RNA interference of YWHAZ gene on the cell proliferation of colon cancer HCT-8 cells,and to elucidate the mechanism.Methods Four siRNA plasmids targeting human YWHAZ gene,including pGPU6/GFP/Neo-YWHAZ-792,pGPU6/GFP/Neo-YWHAZ-733,pGPU6/GFP/Neo-YWHAZ-612,and pGPU6/GFP/Neo-YWHAZ-505, were constructed and regarded as RNA interference plasmid groups. pGPU6/GFP/Neo-shNC plasmid was regarded as negative control plasmid group. RT-PCR was used to detect the expression of YWHAZ mRNA and the inhibitory rates of HCT-8 cells; the proliferation rate of HCT-8 cells was detected by MTT method.Results The transfection rate of cells was 60% under inverted fluorescence microscope;the RT-PCR results showed that the levels of YWHAZ mRNA in RNA interference plasmid groups were lower than those in negative control plasmid group(P<0.01),and the average inhibitory rate was 52.86%. The MTT results showed that compared with negative control plasmid group,the proliferation rates of HCT-8 cells in RNA interference plasmid groups were decreased (P<0.01),and the average proliferation rate was 68.75%.Conclusion Interference of YWHAZ gene might inhibit the transcription of the gene,and further inhibit the proliferation of HCT-8 cells.

Objective To study the effects of fluoride level on the mechanical properties,such as flexural strength,elastic modulus and micro-tensile bond strength(μTBS),of new nano-fluoride-containing resin.Methods Z350 and four composite resins (containing 0%,10%,20% and 30% fluoride)were selected. Five kinds of resins were used to make standard samples with the size of (25.0±2.0)mm×(2.0±0.1)mm×(2.0±0.1)mm,and there were 5 specimens in each group. The flexural strength and elastic modulus of the specimens were assessed using a three-point bending test on a universal test machine. Twenty five human molars were randomly divided into five groups,and there were 5 molars in each group. The teeth were sectioned with a cross-section of (1.0±0.2)mm²,and there were ten specimens in each group,and the μTBS of specimens were detected by Micro Tensile Tester. The fracture modes of specimens were observed under low magnification microscope.Results The flexural test results showed that there was no significant difference of the flexural strength of specimen among all groups(P>0.05);the elastic modulus of specimens in 20%F group was higher than those in the other groups(P<0.05).The micro-tensile test results showed that the μTBS of the specimens in 30%F group was lower than those in 0%F group,10%F group and 20%F group(P<0.05);but there were no significant differences of μTBS between 0%F group and 10%F or 20% F groups (P>0.05).Compared with Z350 group,the μTBS of the specimens in 20% and 30%F groups were significantly decreased (P<0.05),but there were no significant differences of μTBS between 0%, 10% F groups and Z350 group (P>0.05).All specimens were broken from the adhesive surface.Conclusion When the fluoride level is lower than 20%,it has no effect on μTBS;when the fluoride level is above 20%,the μTBS is significantly decreased.

Objective To investigate the effect of miR-18b on the chemosensitivity of human breast cancer MDA-MB-231 cells,and to elucidate its possible mechanism.Methods The breast cancer MDA-MB-231 cells were divided into MDA-MB-231 group,MDA-MB-231+DDP group,MDA-MB-231-NC group,MDA-MB-231-NC+DDP group,MDA-MB-231 mimic group,and MDA-MB-231 mimic+DDP group.The target gene of miR-18b was predicted by bioinformatics method;Dual-luciferase Reporter Assay was used to demonstrate the binding of miR-18b 3' UTR of targets;Western blotting method was used to analyze the expression levels of ATM protein;qRT-PCR method was used to detect the expression levels of miR-18b in MDA-MB-231 cells;CCK-8 method was used to detect the inhibitory rate of growth of MDA-MB-231 cells.Results The Luciferase results showed that the ATM was the target gene of miR-18b-MB-231,which combined with the ATM 3'UTR sequence part;compared with MDA-MB-231 group, the expression levels of miR-18b in the MDA-MB-231 cells in MDA-MB-231 mimic group and MDA-MB-231 mimic+DDP group were increased(P<0.05).The Western blotting results showed that comared with MDA-MB-231 group,the expression level of ATM protein in MDA-MB-231 group was increased(P<0.05).Compared with MDA-MB-231-NC group,the expression level of ATM protein of the cells in MDA-MB-231 mimic group was decreased(P<0.05);compared with MDA-MB-231-NC+DDP group,the expression level of ATM protein of the cells in MDA-MB-231 mimic+DDP group was decreased(P<0.05).The CCK-8 results showed that compared with MDA-MB-231-NC group,after treated with different concentrations of DDP,the inhibitory rates of the proliferation of the cells in MDA-MB-231 mimic group were increased.Conclusion miR-18b can enhance the chemosensitivity of human breast cancer MDA-MB-231 cells to DDP through target-regulating ATM protein.

Objective To explore the effect of temozolomide (TMZ) in combination with metformin (MET) on the elimination of glioblastoma stem cells(GSCs) in vitro,and to clarify the mechanism.Methods The human glioma U87 cells were cultured in the neural stem cell medium,and the GSCs were identified by immunofluorescence methods;the GSCs were selected and were treated with control solution(control group), different concentrations of TMZ(TMZ group)and MET(MET group) or TMZ combined with MET(TMZ+MET group).The number of secondary neurospheres were counted under microscope, and the inhibitory rate of proliferation of GSCs was detected by CCK-8 assay;flow cytometry and Annexin Ⅴ and PI staining flow cytometry were used to detect the percentage of cell cycle and the apoptotic rate of GSCs,respectively.Results Compared with control group, the number of secondary neurospheres in TMZ+MET group was significantly decreased in a concentration-dependent manner;the number of secondary neurosphere in TMZ+MET group was significantly lower than those in TMZ group and MET group(P<0.01).Compared with control group, the inhibitory rates of the proliferation of GSCs in TMZ and MET groups were increased,the inhibitory rate of proliferation of GSCs in TMZ+MET group was higher than those in TMZ group and MET group(P<0.05),and the combination index(CI)<1.The flow cytometry results displayed that the percentage of the cells at G₂/M phase in TMZ+MET group was significantly higher than those in TMZ group and MET group(P<0.05),and the apoptotic rate of GSCs in TMZ+MET group was significantly higher than those in TMZ group and MET group (P<0.05).Conclusion The combination of TMZ and MET can inhibit the serial self-renewal capability of GSCs as well as eliminate GSCs,and its mechanism may be related to the inhibition of the cell cycle progression of GSCs and induction of apoptosis.

Objective To study the therapeutic effect and mechanism of arterial infusion chemotherapy on glioma of the rats,and to provide the basis for clinical application.Methods 24 rats were randomly divided into artery group,vein group,and control group; there were 8 rats in each group.The chemotherapy medicine VM-26 were injected through tail intravenous(vein group) and carotid arteries (artery group) into the rats, respectively,and the rats in control group were given normal saline. The tumor size and survival period of the rats were observed;all the rats were killed after giving different treatment for one week, and the expression levels of proliferating cell nuclear antigen(PCNA) and topoisomeraseⅡ(TOPO Ⅱ) in the tumor cells of the rats were detected by PCR method,meanwhile the number of PCNA positive cells in the tumor cells were detected by immunohistochemical method;the apoptotic rates of the tumor cells of the rats were detected by flow cytometry method.Results After treatment,the tumor growth speed of the rats in artery group was the slowest,which was followed by vein group, and the speed of the rats in control group was the fastest.The tumor size of the rats in artery group was smaller than those in vein and control groups,and the tumor size of the rats in vein group was smaller than that in control group(P<0.05).Compared with control group,the survival period of the rats in artery and vein groups were extended (P<0.05);the survival period of the rats in artery group was longer than that in vein group,but there was no statistically significant difference between two groups (P>0.05).The expression levels of PCNA and TOPO Ⅱ mRNA in the tumor tissue of the rats in artery group were lower than those in vein and control groups (P<0.05),and the expression levels of PCNA and TOPO Ⅱ in the tumor tissue of the rats in vein group were lower than those in control group (P<0.05).The number of PCNA positive cells in the tumor tissue of the the rats in artery group was lower than those in vein group and control group (P<0.05).The apoptotic rate of tumor cells of the rats in artery group was higher than those in vein group and control group(P<0.05).Conclusion The way of arterial perfusion chemotherapy could make the proliferation of tumor cells slow,and promote the apoptosis of tumor cells obviously,and can control the tumor growth more effectively.

Objective To observe the repair effects of neural stem cells(NSCs) combined with erythropoietin (EPO) on transverse spinal cord injury in the adult rats,and to provide a theoretical basis for clinical treatment of spinal cord injury.Methods Forty Wistar rats were used to set up T10 spinal cord transaction models and were randomly divided into control group(n=10),NSCs group(n=10),EPO group(n=10), and NSCs+EPO group(n=10).NF-200 immunohistochemistry and immunofluorescence staining were used to detect the morphology of the regenerating nerve fibers of the rats in various groups 8 weeks after the operation;the BBB score was used to evaluate the restoration of motor function of the hind limbs of the rats in various groups.Results The results of histomorphology showed that the stump of the transected spinal cord tissue atrophied,and there were a large amount of the formation of hole between white matter and gray matter of spinal cord of the rats in control group.The regeneration of nerve fibers could not be seen clearly of the rats in control group.The stump of transected spinal cord tissue atrophied slightly of the rats in NSCs+EPO group,and a large amount of nerve fibers with messy and disorderly state were visible in the cross-sectional area,and there was a lot of continuous nerve fibers getting through the cross-sectional area,and a small number of the formation of hole between white matter and gray matter of spinal cord could be observed.In the experiment,a lot of FITC-conjugated anti-neurofilament antibody NF-200 labelled nerve fibers regenerated in the side of head of the transected area in NSCs+EPO group could be observed,which grew disorderly,and got through the tail side of the damage zone;However,there was a small number of the regeneration of nerve fibers in NSCs group,and these fibers didn't get through the damage zone.The BBB scores of motor function of the hind limbs of the rats in NSCs+EPO group were higher than those in other groups within 7 d after the operation(P<0.05),and the BBB scores of the rats in NSCs group were also higher than those in control group and EPO group(P<0.05).Conclusion NSCs transplantation combined with intraperitoneal injection of EPO could promote the recovery of the neurological function and benefit the survival and regeneration of injured axons after spinal cord injury in the adult rats.

Objective To observe the effect of urinary kallidinogenase on the expressions of insulin-like growth factor 1(IGF-1) and insulin-like growth factor 1 receptor(IGF-1R) after ischemia reperfusion injury in the rats, and to clarify the mechanism of brain protection.Methods 24 adult SD rats were randomly divided into sham operation group(n=8), model group(n=8),and experiment group(n=8).The middle cerebral artery occlusion reperfusion models were made by the suture method for the rats in model and experiment groups.The rats in sham operation group were taken the similar operation without suture.The rats in model group were given urinary kallilidinogenase 5 min after the reperfusion.The expressions of IGF-1 and IGF-1R mRNA and proteins,and the number of IGF-1 and IGF-1R positive cells in the ischemic region of the brain tissue of the rats in various groups were detected by RT-PCR,immunohistochemical,and Western blotting methods.Results Compared with sham operation group,the expression levels of IGF-1 and IGF-1R mRNA and proteinsand the number of positive cells in the brain tissue of the rats in model group and experiment group were increased (P<0.05);compared with model group,the expression levels of IGF-1 and IGF-1R mRNA and proteins and the number of positive cells in the brain tissue of the rats in experimental group were increased(P<0.05).Conclusion Urinary kallidinogenase can improve the cerebral ischemia reperfusion,which is associated with the promotion of the expressions of IGF-1 and IGF-1R.

Objective To study the effect of Ginkgo biloba extract (GBE) on the adipogenic differentiation ability of the bone marrow mesenchymal stem cells(BMMSCs),and to clarify its significance in treatment and prevention of osteoporosis at the cell level.Methods The third generation of BMMSCs were cultured with adipogenic induction solution(control group),and 50,100,150,200, and 400 mg·L^-1 GBE(50,100,150,200, and 400 mg·L^-1 GBE groups) for 7 d.The morphology of BMMSCs and the expressions of surface antisgens CD44,CD105, and CD34 were observed;the formation of lipid droplet in the BMMSCs was observed by Oil Red O staining;the quantitative analysis of the adipogenic ability of the BMMSCs was performed;RT-PCR method was used to test the expression levels of fatty acid-binding protein(AP2) and peroxisome proliferator activated receptor-γ(PPAR-γ) mRNA.Results The morphological detection results showed that the BMMSCs were spindle shaped after cultured in vitro for 3 d,and grew in whorls and arranged in the same direction after cultured for 7 d.The surface antigens CD44 and CD105 showed positive expression,and CD34 showed negative expression,which proved the BMMSCs were extracted correctly;the Oil red O staining results showed a large of lipid droplets which was seen under microscope,and the number of lipid droplets was reduced with the increasing of the concentrations of GBE; compared with control group,the A₅₁₀ values of the BMMSCs in other groups were decreased(P<0.05).The RT-PCR results showed the expression levels of AP2 and PPAR-γ mRNA were decreasd in other groups compared with control group (P<0.05).Conclusion GBE can inhibit the adipogenic differentiation ability of BMMSCs in vitro,and the inhibitory ability is enhanced with the increasing of GBE concentration.

Objective To detect the expressions of Cks1,Skp2, and p27^kip1 proteins in tongue squamous cell carcinoma tissue and to analyze their associations with the clinicopathological characteristics of the patients with tongue squamous cell carcinoma, and to clarify their roles in the development and prognosis of tongue squamous cell carcinoma.Methods The expressions of Cks1,Skp2 and p27^kip1 proteins in 45 cases of human tongue squamous cell carcinoma tissue,30 cases of adjacent carcinoma tissue and 10 cases of normal tongue mucosa tissue were detected by immunohistochemistry;the differences of the positive expression rates of Cks1,Skp2,and p27^kip1 proteins in different kinds of tongue tissues were compared; the relationships between the expressions of Cks1,Skp2 and p27^kip1 proteins with the clinicopathological parameters of the patients were analyzed.Results The expression levels of Cks1 and Skp2 proteins in tongue carcinoma tissue were obviously increased and the positive expression rate of Cks1 and Skp2 proteins were 73.3% and 62.2%.The expression levels of Cks1 and Skp2 proteins in the adjacent carcinoma tissue and normal tongue mucosa tissue were decreased gradually,and the positive expression rates of Cks1 and Skp2 proteins were 23.3%,10.0% and 36.7%,10.0%, respectively.The expression level of p27^kip1 protein in the tongue carcinoma tissue was decreased or loss, and the positive expression rate of p27^kip1 protein was 24.4%.The expression levels of p27^kip1 proten in the adjacent carcinoma tissue and normal tongue mucosa tissue were increased gradually,and the positive expression rates of p27^kip1 protein were 30.0% and 70.0%,respectively.The postive expression rates of Cks1 and Skp2 proteins in the tongue carcinoma tissue were higher than those in the adjacent carcinoma tissue and normal tongue mucosa tissue(P<0.05),and the positive expression rate of p27^kip1 protein was lower than those in the adjacent carcinoma tissue and normal tongue mucosa tissue (P<0.05).The positive expression rates of Cks1,Skp2 and p27^kip1 proteins in the tongue carcinoma tissue were significantly correlated with the lymph node metastasis,TNM stages,differentiation degrees,and clinical stages(P<0.05);but they were not correlated with gender and age (P>0.05).Conclusion The positive expression rates of Cks1,Skp2 proteins in tongue carcinoma tissue are increased and the positive expression rate of p27^kip1 protein is decreased;the detection of the positive expression rates of Cks1,Skp2 and p27^kip1 proteins contributes to judging the malignancy grade and prognosis of the tongue sequamous cell carcinoma.

Objective To detect the level of serum procalcitonin(PCT) in the patients with community acquired pneumonia(CAP),and to clarify its significance in diagnosis and treatment of CAP.Methods 127 CAP patients and 30 cases of normal individuals were selected. According to the sputum bacterial culture results,127 CAP patients were divided into positive bacteria culture group (n=54)and negative bacteria culture group (n=73);according to the diagnostic criteria of severe pneumonia,the bacteria culture positive group was divided into severe pneumonia group (n=13) and non-severe pneumonia group(n=41). The levels of serum PCT,the white blood cell (WBC) count,neutrophil percentage(NEUT%) and levels of C-reactive protein (CRP) of the objects were detected by double antibody sandwich method.Results After treated for 8 d,the levels of serum PCT of the objects in positive bacteria culture group and negative bacteria culture group and normal control group had no significant differences. After treated for 8 d, the levels of serum PCT of the objects in positive bacteria culture group and negative bacteria culture group were lower than that of the objects on the first day after treatment.The level of serum PCT of the patients in severe pneumonia group was significantly higher than that in non-severe pneumonia group;the WBC count,NEUT% and CRP level of the patients in severe pneumonia group were higher than those in non-severe pneumonia group,but the difference was not statistically significant. When consindering the bacterial culture as the gold standard for diagnosis of bacterial infection,the sensitivity and specificity of the serum level in diagnosis of bacterial CAP were superior to NEUT% and WBC count.Conclusion The level of serum PCT plays a role in the diagnosis of CAP,and it could help to judge whether the CAP patient gets a bacterial infection and assess the severity of pneumonia and the discontinuation of antibiotics.

Objective To investigate the expressions of silent information regulator 1 (Sirt-1) and hypoxia-inducible factor 1α (Hif-1α) in peripheral blood mononuclear cells (PBMCs) of the chronic obstructive pulmonary disease (COPD) patients,and to clarify their potential roles in the occurrence and development of COPD.Methods 42 patients with COPD in acute exacerbation stage (n=20) and in stable stage (n=22) and 20 healthy people were chosen.The lung function indexes of all cases were examined by lung function machine.The expressions of Sirt-1 and Hif-1α mRNA and protein in PBMCs of the objects in various groups were measured by Real-time PCR and Western blotting method. The relationships between the expression levels of Sirt-1 and Hif-1α and lung function were analyzed.Results Compared with control group,the calculated ratios of forced expiration volume in one second to predicted volume (FEV₁%) and the the calculated ratios of forced expiration volume in one second to forced vital capacity (FEV₁/FVC%) of the patients with COPD in stable stage and acute exacerbation stage were significantly decreased (P<0.05);compared with the patients in stable stage of COPD group,the FEV₁ and FEV₁/FVC% of the patients with COPD in acute exacerbation stage group were decreased (P<0.05).The expression levels of Sirt-1 mRNA and protein of the patients with COPD in acute exacerbation stage and stable stage were significantly lower than those in control group (P<0.05 or P<0.01);compared with the patients in stable stage of COPD group,the expressions of Sirt-1 mRNA and protein in PBMCs of the patients with COPD in acute exacerbation stage group were decreased(P<0.05);meanwhile,the expression levels of Hif-1α mRNA and protein of COPD patients were significantly higher than those in control group (P<0.05, P<0.01),especially in acute exacerbation stage(P<0.01);compared with the patients in stable stage of COPD group,the expressions of Hif-1α mRNA and protein in PBMSCs of the patients with COPD in acute exacerbation stage group were increased(P<0.05).The Pearson correlation analysis results showed that there were positive correlations between the expression levels of Sirt-1 mRNA (protein) and the FEV₁%(r_mRNA=0.661,r_protein=0.545) and FEV₁/FVC (r_mRNA=0.513,r_protein=0.491)(P<0.05),and there were negative correlations between the expression of Hif-1α mRNA (pretein) and FEV₁%(r_mRNA=-0.812,r_protein=-0.781) and FEV₁/FVC(r_mRNA=-0.661,r_protein=-0.674) (P<0.05).Conclusion The expression level of Sirt-1 gene is decreased and the expression level of Hif-1α gene is increased in the patients with COPD,which may be associated with the incidence and pathogenesis of COPD.

Objective To investigate the expressions of CD34 and CD38 antigens in the leukemia cells in the patients with firstly-diagnosed acute myeloid leukemia (AML) and their relationships with the prognosis,and to explore the role of the expressions of CD34 and CD38 antigens in predicting the prognosis of the AML patients.Methods The expressions of CD34 and CD38 antigens in the leukemia cells in 94 cases of AML patients were detected by flow cytometry. 36 patients with CD34⁺ CD38^- antigen and 58 patients with CD34⁺CD38⁺ antigen were identified by the expressions of CD38 and treated with IA Regimen. The complete remission rate (CRR),the relapse rate,the median overall survival (OS),the median disease free survival (DFS) and the survival rate of the patients in two groups were compared.Results There was no significant difference of the CRR of the patients between CD34⁺ CD38^- and CD34⁺CD38⁺ groups (77.8% vs 86.2%,P>0.05). However,the relapse rate and the 1-year relapse rate of the patients with CD34⁺ CD38^- antigen (53.8% and 36.0%) were higher than those of the patients with CD34⁺CD38⁺ antigen(27.0% and 14.3%)(P<0.05).The median OS and median DFS of the patients in CD34⁺ CD38^- group (13.60 months and 12.87 months) were shorter than those in CD34⁺CD38⁺ group (20.33 months and 33.93 months) (P<0.05).The 1-year and 2-year survival rates of the patients in CD34⁺ CD38^- group (52.8% and 38.9%) were lower than those in CD34⁺CD38⁺ group (75.9% and 48.3%)(P<0.05).Conclusion The expression of CD34⁺ CD38^- antigen in the leukemia cells offers a new prognostic factor for the AML patients. The expression of CD34⁺ CD38^- antigen predicts a worse prognosis in the AML patients.

Objective To investigate the associations between single nucleotide polymorphisms(SNPs)of DNA methyltransferase 3b(DNMT3b) gene and the risk of gastric cancer in the Han population from North China,and to clarify whether DNMT3b gene is the susceptible gene of gastric cancer.Methods 447 patients with gastric cancer were selected as case group and 961 healthy controls were used as control group.The distribution of genotypic frequencies of five SNPs(rs6119954,rs1569686,rs4911107,rs4911259,and rs8118663)in two groups were detected by TaqMan assay.The expression levels of DNMT3b protein in 104 cases of cancer tissue from the patients with gastric cancer and 85 cases of tumor-adjacent tissue from the patients with gastric cancer were analyzed by immunohistochemistry method.Results The distributions of genotypic frequencies of five SNPs in case group and control group were similar.No significant association was found between the five SNPs and the risk of gastric cancer(P>0.05).Compared with the tumor-adjacent tissue,the expression level of DNMT3b protein in the gastric cancer tissue was increased(P<0.05).There were no siginificant differences between the genotypes of the five SNPs and the expression levels of DNMT3b protein(P>0.05).Conclusion There is no association between the polymorphisms of DNMT3b and the occurrence of gastric cancer in Han population from North China.DNMT3b gene may not be the susceptible gene of gastric cancer.

Objective To investigate the changes of heart rate turbulence (HRT) in the patients with heart failure,and to clarify its clinical significance in the prognosis assessment of the patients with heart failure.Methods 50 patients with chronic heart failure(CHF) (CHF group) and 25 normal controls (control groups) were selected. The levels of serum brain natriuretic peptide (BNP) within 24 h after admission in CHF patients were detected,and the values of left ventricular ejection fraction(LVEF)and left ventricular end diastolic diameter (LVEDD) of all the 75 subjects were measured by echocardiography; the values of turbulence onset(TO) and turbulence slope (TS) were obtained by Holter analysis. The values of TO,TS,LVEF,LVEDD,levels of BNP,and HRT grading were compared between CHF group and control group.And the major cardiovascular events,mortality and repeated episodes of the hospitalization for the heart failure symptoms after discharge were recorded; Logisitic regression analysis was used to analyze the relationship between the death and HRT grading.Results The value of TO and value of LVEDD of the patients in CHF groups were significantly higher than those in control group(P<0.01),and the value of TS and value of LVEF were significantly lower than those in control group(P<0.01).The value of TO,level of BNP,and value of LVEDD of the patients in moderate and severe heart failure group were slightly higher than those in mild heart failure group(P<0.05,P<0.01),and the value of TS and value of LVEF were significantly lower than those in mild heart failure group(P<0.01).The value of TS of the patients with good prognosis was significantly higher than that of the patients with poor prognosis(P<0.05),and the value of TO was significantly lower(P<0.05). There were positive relationship between the value of TO and level of BNP and value of LVEDD(r=0.33,r=0.349,P<0.05) and negative relationship with value of LVEF(r=-0.440,P<0.05).There were positive relationship between the value of TS and value of LVEF(r=0.326,P<0.05)and negative relatiohship with level of BNP(r=-0.403,P<0.05). The value of TO and level of BNP of the patients in mortality group were significantly higher than those in survival group(P<0.05),and the values of TS and LVEF were significantly lower than those in control group(P<0.05).Conclusion HRT significantly is reduced in the CHF patients,and the decreasing of HRT has the prognostic significance in the CHF patients.

Objective To explore the effects of tumor necrosis factor α(TNF-α) signal pathway related proteins in the serum of the asthma children in the pathogenesis of asthma,and to provide the basis for prevention and treatment of asthma in the children.Methods The serum of normal children(control group,n=31) and asthma children(asthma group,n=33) in clinical remission,chronic persistent and acute exacerbation periods were obtained. The expression levels of TNF-α,cysteine-containing aspirate-specific protease-8(Caspase-8),TNF receptor-associated factors-2 (TRAF-2),reportor interacting protein (RIP) and cysteine-containing aspirate-specific protease-3 (Caspase-3) proteins were detected by ELISA assay.Results The expression levels of TNF-α protein in serum of the asthma children in clinical remission group,chronic persistent group,and acute exacerbation group were increased. Compared with control group,the expression levels of TNF-α, Caspase-8,and Caspase-3 proteins in serum of the asthma children in acute exacerbation group were significantly increased(P<0.05);the expression level of Caspase-3 protein in serum of the asthma children in clinical remission group was significantly decreased(P<0.05);but the expression level of Caspase-3 protein in serum of the asthma children in chronic persistent group was significantly increased(P<0.05).There were no significant differences of the expression levels of TRAF2 and RIP proteins between asthma group and control group(P>0.05).Conclusion TNF-α signal pathway is closely related to the development of asthma in children,and its mechanism may be activating the TNF-α pathway to promote the expression levels of Caspase-3 and Caspase-8 in the cells and to induce asthma.

Objective To explore the expression levels of ERAP1 gene in the peripheral blood of the essential hypertension patients, and to identify the single nucleotide polymorphisms(SNPs) in 3'UTR of ERAP1 gene associated with essential hypertension.Methods 300 patients with hypertension (hypertension group) and 233 normal controls (control group) were recruited to conduct a case-control study in Jilin Han population. The expression levels of ERAP1 in the peripheral blood of the objects were detected by ELISA method;the SNPs in 3' UTR of ERAP1 gene were identified by pool-sequencing method;the genotyping was performed for each participant by PCR sequencing method.Results The expression levels of ERAP1 in the peripheral blood of the patients in hypertension group were significantly lower than those in control group (P<0.05). Two SNPs,E20-790G >A and E20-816C >T,were identified in 3'UTR of ERAP1 gene. The distribution of the genotypic and allelic frequencies of E20-816C >T polymorphism was significantly different between hypertension group and control group (P<0.05). The Logistic regression results showed that adjusting for the confounding factors,the genotypic frequency of CT gene in hypertension group was decreased significantly(P<0.05,OR=0.145,95% CI:0.031-0.675).Conclusion E20-816C>T polymorphism may be related to essential hypertension in the Jilin Han population,and the decreasing genotypic frequency of CT gene may be related to the down-regulated expression of ERAP1.

Objective To explore the correlation between the daily mean temperature (DMT) and the hospital admissions of the patients with acute exacerbation of chronic obstructive pulmonary disease(AECOPD) in different seasons in Changchun City,and to clarify the effect of DMT on AECOPD.Methods The clinical data of 2 564 AECOPD patients and meteorological variables in the same period were collected. A case-crossover study design was used to investigate the association between different meteorological variables and AECOPD in different seasons.Results Under the conditions of adjusting the influences of the daily mean relative humidity (DMRH),daily mean wind speed (DMWS),and daily mean atmospheric pressure (DMAP),when the DMT was increased by 1%,the hospital admissions of the patients with AECOPD in spring and summer were increased by 18.9% (OR=1.189,95%CI=1.152-1.228) and 14.2% (OR=1.142,95%CI=1.074-1.214) (P<0.05),and it was decreased by 9.7%(OR=0.903,95%CI=0.874-0.933) in autumn (P<0.05). But there was no significant association between the DMT and the hospital admissions of the patients with AECOPD in winter (P>0.05).Conclusion The increasing of DMT may be a risk factor of the patients with AECOPD in spring and summer;however,it may be a preventive factor in autumn and winter.

Objective To investigate the association between rs290487 polymorphisms of transcription factor 7 like 2 (TCF7L2) gene and type 2 diabetes mellitus (T2DM) in Ningxia Hui population,and to clarify whether the TCF7L2 gene is the susceptible gene of T2DM.Methods Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method was used to detect the distribution of the geotypic and allelic frequencies of rs290487 site of TCF7L2 gene of the objects,including 111 T2DM patients (T2DM group) and 109 healthy individuals (control group)in Ningxia Hui population. χ² test was used to compare the differences of the allelic frequency and genotypic frequcecy of the objects between two groups.Results Three genotyps TT,CT,and CC were found both in T2DM group and control group; the genotypic frenquencies of rs290487 site of TCF7L2 gene did not deviate from the Hardy-Weinberg equilibrium in two groups (P>0.05); there was no significant difference of the genotypic frequency of rs290487 site between two groups(χ²=5.370,P>0.05);there was no significant difference of the alleclic frequency of rs290487 site between two groups (χ²=0.034,P>0.05).Conclusion The polymorphisms of rs290487 site of TCF7L2 gene has no relationship with T2DM in Ningxia Hui population,and TCF7L2 gene may be not the susceptible gene of T2DM.

Objective To explore the therapeutic effect and advantages of small incision releasing of transverse carpal ligament in the treatment of mild and moderate carpal tunnel syndrome(CTS),and to clarify its special clinical curative effect.Methods An incision 2.0 cm in length was made at the level of the middle wrist crease and the palmaris longus tendon was on the midpoint of the incision,as an anatomical landmark. After the skin and subcutaneous tissue were cut,the palmaris longus tendon and the median nerve were exposured,and the transverse carpal ligament proximal part was revealed.A mosquito haemostat was advanced forward between palmar aponeurosis and flexor retinaculum and the proximal portion of transverse carpal ligament was visualized. The palmar aponeurosis and skin were lift up and the wrist joints was extend about 15°. The wrist transverse ligament was completely cut in the direct vision along the line of the ulnar side of the median nerve ring axial extension cord by the blunt organization scissor.Results The follow-up results showed that all the clinical symptoms of all the patients were released obviously,and the latent periods of sensory nerver action potential (SNAP) of the median nerve at the wrist were shortened when the index and middle fingers were stimulated 2 weeks after the operation;3 months after the operation,the symptoms of numbess and pain disappeared in all cases and the two-point discrimation of the index finger pulp had a recovery to the level of (5.0±0.5) mm,and the muscle strength of the abductor pollicis brevis muscles was partly recovered,and the latent periods and amplitudes of the abductor pollicis brevis compound muscle action potential (CMAP) became shortned and increased,respectively.One year after the operation,all the patients had no clinical symptoms of numbess and pain.The function of thumb on the plam got normal. All the electrophysiologic parameters turned to the normal levels and no scar discomfort and other apalmar arch were seen.Conclusion Release of the transverse carpal ligament under the direct vision by a small incison is an effective surgical procedure for CTS.

Objective To investigate the value of ¹⁸F-FDG PET/CT imaging in the diagnosis and differential diagnosis of the primary central nervous system lymphoma (PCNSL),and to illustrate the characteristics of imaging findings,and to provide the reference and basis for clinical treatment.Methods A retrospective analysis of 55 cases of ¹⁸F-FDG PET/CT image data and immunohistochemical results including 8 cases of PCNSL,19 cases of gliomas and 28 patients with cerebral metastatic carcinoma confirmed by pathology was performed;the maximum standardized uptake value (SUV_max) was compared between three kinds of diseases.Results PCNSL ¹⁸F-FDG PET/CT performed as an abnormal radioactivity uptake showed strong together as nodular,crumb,bunch of ribbon,and the surrounding tissue boundary was not clear and the edema was rare.The radioactivity uptake of glioma in ¹⁸F-FDG PET/CT imaging depended on the pathological types,and the uptake of fluoredeoxyglucose(FDG) of generally low grade glioma was reduced,and the SUV_max was 2.2-4.3;the uptake of FDG of high grade glioma was increased,and the SUV_max was 9.3-17.2;the uptake of tracer in lesion was not homogeneous,and the flake edema could be seen around the lesions.Typical cerebral metastatic carcinoma performed as the increasing uptake of the radioactivity lesions, and the flake edema could be seen around the lesions. The SUV_max of PCNSL patients was higher than that of cerebral metastatic carcinoma patients (P<0.05);there was no statistically significant difference of SUV_max beweteen PCNSL and glioma patients(P>0.05).Conclusion The ¹⁸F- FDG PET/CT imaging of PCNSL patients has certain imaging characteristics,which can be used for differential diagnosis with other central nervous system tumors.

Objective To understand the levels of Mn,Fe,Zn,Ca,and Mg elements in the hair of the adults, and to discuss their correlations with the body mass index(BMI).Methods A total of 657 adults,which were sampled through random cluster sampling method,were enrolled by questionnaire survey,physical examination,and hair sample collection. The levels of Mn,Fe,Zn,Ca,and Mg elements in the hair were detected by ICP-MS method.Results There were siginficant differences of the levels of Mn,Fe,Zn,Ca,and Mg elements in hair of the adults with different genders and ages(P<0.05).322(49.0%)adults exceeded the BMI standard in 657 adults. There were no significant differences of the BMI and BMI exceeded rates between the adults with different genders(P>0.05).There were significant differences of the BMI and BMI exceeded rates between the adults with different ages (P<0.05).The BMI exceeded rate was high in the adults aged 40-69 years,and it reached the highest in the adults aged 50-59 years. The levels of five kinds of mineral elements in the BMI exceeded adults were lower than those with normal BMI(P<0.05).After age and gender were controlled,the partial correlation analysis results showed BMI had negative correlation with Mn(r_s=-0.117,P=0.003),Fe(r_s=-0.114,P=0.004),Ca(r_s=-0.108,P=0.006) and Mg(r_s=-0.089,P=0.023) elements; and had no correlations with Zn element (r_s=0.045,P=0.247).Conclusion Age and gender are the potential influencing factors for the levels of mineral elements in human.The levels of Mn,Fe,Zn,Ca,and Mg elements of the BMI exceeded adults are decreased significantly. The BMI exceeded adults should pay attention to the intake of mineral elements reasonably.

Objective To understand the hospital visiting situation and disease burden and to analyze the influencing factors of disease burden of the patients with foodborne diseases in Jilin Province,and to provide the basis for prevention and control of foodborne diseases.Methods 7 353 people (3 065 for 2012 year and 4 288 for 2013 year) lived for 6 consecutive months and above from 2012 to 2013 were selected from ten monitoring areas of Jilin Province by multi-stage stratified random sampling method, and the data of these residents were collected with questionnaire by face-to-face interview. The hospital visiting and disease burden situation of the patients with different kinds of foodborne diseases were analyzed; the influencing factors of disease burden of the patients with foodborne diseases were detected by univariate and multiple linear regression analysis.Results The rate of hospital visiting of the patients with foodborne diseases was 13.10%,and there were significant differences of the rates of hospital visiting of the patients with different ages,culture degrees,numbers of acute gastroenteritis over the past 4 weeks and disease durations(P<0.05). The rate of hospital visiting in the patients of 35-44 age group was higher than those in other groups(P<0.05);the rates of hospital visiting of the patients with university and above degree,occurring 2 times and above over the past 4 weeks or illness lasting 4 d and above were higher than those in other groups(P<0.05). The total economic burden and per capita economic burden of 157 patients with foodborne diseases were 5 847.82 and 37.25 yuan.The first hospital level and disease duration were the main influencing factors of economic burden of foodborne diseases.Conclusion The rate of hospital visiting of the patients with foodborne diseases is not high,but it still brings certain economic burden in the patients.It is important to improve the rate of capacity utilization and the treatment of primary health care institutions to reduce the economic burden of the patients with foodborne diseases.

Objective To observe the distribution of gonion (Go) on the frontal photograph of the normal young adult,and to hypothesize a new facial reference line for locating gonion,and to provide the basis for studing the effect of orthodontic treatment on the frontal view of facial soft tissue.Methods 108 normal young adults were selected. Each subject was taken two frontal photographs under natural head position,such as a standardized one (A) and the other one with marked Go by facebow (B). The Zy-Me segment was used as a new reference line and the distribution of Go towards the Zy-Me segment was observed. The true value and calculated value of Zy-Zy/Go-Go were compared to prove the validity of the new method. The differences of Zy-Me and Zy-Zy/Go-Go between different genders were also analyzed.Results By making a perpendicular towards the Zy-Me segment via Go,it showed that the feet were located near the midpoint of the segment (50.31±2.21). The distribution of the frequency of Zy-H/Zy-Me followed the normal distribution (Normality=0.990). There were no significant differences between the true values and the calculated values of Zy-Zy/Go-Go (P>0.05, Cronbach's α coefficient > 0.9). There were no significant differences of Zy-H/Zy-Me and Zy-Zy/Go-Go of the objects between different genders(P>0.05).Conclusion On the frontal photograph of the normal young adults,the projected point of Go locates near the midpoint of the Zy-Me segment. The intersection of the facial contour and the perpendicular towards the midpoint of Zy-Me segment can be seen as Go in clinical trials.

Objective To select the best extraction solvent of the raw polysaccharide in northeast water chestnut,and to compare the purify effects of 4 kinds of purification methods for raw polysaccharide.Methods The raw extracts from northeast water chestnut were divided into three parts. The raw polysaccharide in northeast water chestnut was extracted with methanol,ethanol,and acetone.The polysaccharide of water-chestnut was deproteinized by Sevag,trichloroacetic acid,hydrochloric,and activated carbon methods. The levels of polysaccharide and protein were detected by colorimetric method,and their purification effects were compared.Results The highest level of the polysaccharide in the water-chestnut extracts was obtained by using acetone as the extraction solvent; the level of polysaccharide was 33.69% and the level of protein was 5.51%.After treated by Sevag,trichloroacetic acid,hydrochloric,and activated carbon,the levels of polysaccharides were 46.53%,43.44%,41.24%,and 36.66%;the levels of proteins were 5.64%,5.41%,5.41%,and 5.51%,respectively.Conclusion Acetone is the best extraction solvent of the raw polysaccharide of northeast water chestnut.The four purification methods have advantages and disadvantages,and the highest polysaccharide level is obtained by Sevag method,and it is suitable for extraction of raw polysaccharide in northeast water chestnut.