To investigate the dynamic expressions of urotensinⅡ (UⅡ) and transforming growth factor-β1 (TGF-β1) in renal tubular epithelial cells and glomeruli of diabetic rats and their roles in pathogenesis of diabetic nephropathy (DN). Methods Thirty male Wistar rats were randomly divided into normal control,2-week (2-wk DM),4-week (4-wk DM),8-week (8-wk DM) and 12-week (12-wk DM) diabetic groups.Diabetic model was induced by a single intraperitoneal injection of streptozotocin (55 mg•kg^-1).Blood glucose (BG),serum and urinary creatinine,urinary albumin (UAlb) and urinary N-acetyl-B-D-glucosaminidase (NAG) contents were dertemined with biochemical methods.Immunohistochemistry was used to detect the expressions of UⅡ,TGF-β1,fibronectin (FN) and collagenⅣ (Col Ⅳ) in the kidney. Results BG,creatinine clearance (Ccr),UAlb and NAG levels were significantly elevated in diabetic groups (P<0.05).As compared with control,positive staining of UⅡ and TGF-β1 was observed in the renal tubular epithelial cells and glomeruli of diabetic rats.Progressively increased UⅡ and TGF-β1 expressions were found in 4-,8- and 12-wk DM groups (P<0.05) with diabetic course compared with 2-wk DM.The expression of UⅡ protein in the renal tubular epithelial cells was positively correlated with urinary NAG content (r=0.895，P<0.01).The expression level of UⅡ in the renal tubular epithelial cells was positively correlated with that of TGF-β1 (r=0.769，P<0.01).The expressions of FN and Col Ⅳ in diabetic kidney in 8- and 12-wk DM groups were obviously increased compared with normal control (P<0.05).Conclusion The expressions of UⅡand TGF-β1 proteins are markedly increased in the renal tubular epithelial cells and glomeruli of diabetic rats,suggesting that UⅡand TGF-β1 may play roles in the renal tubular lesion and extracellular matrix accumulation of diabetic nephropathy.

To analyze the relationship between leukocyte function-associated antigen-1 (LFA-1) mRNA expression and corneal inflammation and discuss the inhibition reaction of nuclear factor-κB（NF-κB）inhibitor on LFA-1 mRNA expression. Methods The corneal suture or combined with subconjunctival injection models were constructed in BALB/C mice.The corneas of each group were excised 1,3,7 and 14 d after operation and LFA-1 mRNA expressions were investigated by reverse transcription polymerase chain reaction(RT-PCR). Results At 1,3 and 7 d after operation, the expressions of corneal LFA-1 mRNA in corneal suture combined with lipopolysaccharide (LPS) (corneal suture+LPs)subconjunctival injection group were higher than those in corneal suture group (P<0.05); at 14 d after operation, there was no significant difference of the expression of corneal LFA-1 mRNA between two groups (P>0.05).At 1 and 7 d after operation, compared with corneal suture+LPS group ,the expression of corneal LFA-1 mRNA increased,but it decreased at 3 and 14 d after operation in the group of corneal suture combined with LPS and pyrrolidine dithiocarbamate (PDTC) (P<0.05). Conclusion A high expression of LFA-1 mRNA can be induced by the corneal stutre combined with LPS subconjunctival injection.The inhibitor of NF-κB (PDTC) can inhibit the expression of corneal LFA-1 mRNA.The expression of LFA-1 mRNA can be blocked by PDTC.

To observe the suppression of special shRNA producing plasmid to hepatitis B virus(HBV) S gene and C gene on HBV replication and expression in HepG2.2.15 cells.Methods pSilenceCircle-U6 including pol Ⅲ promoter was used to construct HBV special shRNA producing plasmid as SC-S and SC-C.The experimental groups included SC-S group,SC-C group,unrelated control SC-N group and blank control group.With different dosages and at different time,shRNA producing plasmid was transfected into HepG2.2.15 cells.HBeAg and HBsAg in the culture media was detected by ELISA assay and HBV DNA in the culture media was measured by dot blotting assay. Results The recombinant shRNA producing plasmid with target sequence was constructed successfully.The inhibitory rates of HBeAg and HBsAg expressions by SC-S were much higher than those by SC-C.The inhibitory effects of HBeAg and HBsAg expressions were increased when the dose of SC-S was greater.The inhibitory effects of HBeAg and HBsAg expressions by SC-S were significant on the 3rd day after transfection and the inhibitory effect was the strongest on the 6th day.The inhibitory rate was still higher on the 9th day after transfection.Dot blotting assay showed the inhibitory effect of HBV replication by SC-S was greater than that by SC-C. Conclusion The shRNA producing plasmid with HBV S gene and C gene can be highly effective to inhibit the replication and expression of HBV.

To produce hybridoma cell lines of monoclonal antibodies(McAb) against recombinant human interleukin-12(rhIL-12) by purified rhIL-12 and identify the specificity of anti-rhIL-12 McAb,and provide a valuable tool for the diagnose and treatment of tumor and immune associated disease .Methods BALB/C mice were immunized with purified rhIL-12p70 and hybridomas were generated with traditional technique.McAb were screened by ELISA with limited dilution and subcloning approach and purified with clutathione sepharose 4B affinity chromatography column.The subtype and titers in the ascites and specificity of McAb were identified by kit and Western blotting respectively.Results Three cell lines of hybridoma constantly secreted McAbs against rhIL-12p70.The chromosomal number of three strains were about 100 and cell lines of hybridoma had been confirmed.Two of them belonged to IgG1 and one of them belonged to IgG2a,Kappa isotypes.Ascites titers were 1∶5.9×106,1∶2.9×10⁷, and 1∶3.6×10⁷.The affinity were EM5>EM6>EV9 in proper order.Conclusion Three hybridoma cell strains are constructed successfully which stably secrete high-titer and high-specific McAb against rhIL-12.

To investigate the protective effect of penehyclidine hydrochloride pretreatment on focal cerebral ischemia/reperfusion injury and expression of caspase-3 in rats.Methods Sixty male Sprague-Dawley rats weighing 250-300 g were randomly divided into 3 groups(n=20): control （C） group in which sham operation was performed; ischemia-reperfusion （I-R）group, in which saline （2.0 mg•kg^-1）was administered for intraperitoneal injection 30 min before anesthesia,then the ischemia model was made by occlusion of the left middle cerebral artery for 120 min; penehyclidine hydrochloride pretreatment （P） group, in which penehyclidine hydrochloride（2.0 mg•kg^-1）was administered for intraperitoneal injection 30 min before anesthesia,then the same 120 min focal cerebral ischemia was performed as described in I-R group.At the end of 24 h reperfusion the animals were decapitated and the brains were removed.The volume of cerebral infarction was detected with TTC staining;Coronal sections including hippocampal tissue were obtained for HE staining.The levels of caspase-3 mRNA expression in hippocampus were detected by using semi-quantitative RT-PCR technique. Results Compared with group I-R，the volume of cerebral infarction in group P were reduced significantly（P< 0.05）;In group P,edema,degeneration and necrosis of nerve cells in the hippocampus of the rats were significantly decreased; The expressions of caspase-3 mRNA in group I-R and group P increased significantly compared with group C（P< 0.05）,and the caspase-3 mRNA expression in group P was lower than that in group I-R（P< 0.05）. Conclusion Penehyclidine hydrochloride has brain protective effect on focal cerebral ischemia/reperfusion injury in rats and its mechanism is related to inhibiting the expression of caspase-3 mRNA in hippocampus.

To analyze the changes of cytotoxicity mediated by NK cells after 24 h coculture with K562 cells and the molecular mechanism. Methods The expressions of MICA/MICB on K562 cells, KIR2DL1, KIR3DL1, NKG2D on NK cells were analyzed by flow cytometry. Cytotoxicity was detected by LDH releasing assay. Results Before editing,the expression rate of MICA/MICB on K562 cells was (79.90±0.87)%, the expression rates of NKG2D,KIRZDL1, and KIR3DL1 on NK cells were (98.27±0.67)%, (45.70±1.22)% and (35.60±1.35)%,respectively.After editing,the expression rates of M1CA/M1CB on K562 cells and NKG2D on NK cells were (35.56±1.01)% and (34.67±3.88)%,respectively;compared with before editing,there was significant difference(P=0.000).The expression rates of KIR2DL1 and KIR3DL1 on NK cells were (47.20±1.08)% and (34.03±1.20)%,compared with before editing,there was no signficant difference(P>0.05).The cytotoxicities against K562 cells by edited NK cells and against the edited K562 cells by NK cells were(24.07± 0.58)% and (16.95±2.00)% at an effector-to-target (E/T) ratio of 20∶1, both were lower than the cytotoxicity against K562 cells by NK cells (54.03%±3.46%), the differences weresignificant (P=0.000). Conclusion Reciprocal immunoediting between NK cells and K562 cells induced by 24 h coculture leads to decreased cytotoxicity of NK cells. The potential molecular mechanism is the changes of NKG2D and MICA/MICB expressions.

To study the isolation,cultivation of human mesenchymal stem cells (MSCs) and the differentiation into neuron-like cells induced by the supernatant of glioma cells in vitro.Methods MSCs were separated from human bone marrow and expanded in vitro and induced by the supernatant of glioma cells.The morphological changes of MSCs after induction were obeserved. The expressions of neuron specific enolase (NSE),neurofilm entprotein (NF) and glial fibrillary acidic protein (GFAP) were detected by immunohistochemistry.Results After induction for 24 h,the differentiated cells showed typical neuronal appearance,and expressed neuron-specific markers,such as NF and NSE.But glial astrocyte marker GFAP was negative.Quantitative count analysis showed that the NSE positive rate of the cells in the induced group (79.5%±3.2%) was much higher than that in the control group (12.1%±2.0%,P<0.01).The NF positive rate of cells in the induced group was 41.2%±2.4%,and the cells in the control group did not express NF.Conclusion The supernatant of glioma cells has inductive effect on differentiation of MSCs into neuron-like cells in vitro.

To obtain the cDNA sequence encoding human α₁-microglobulin/bikunin precursor (AMBP),and construct its recombinant cloning vector. Methods RNA was extracted from human embryo tissues of liver,kidney and pancreas with Trizol reagent.The sequence of human AMBP cDNA was cloned by RT-RCR from those RNAs,and inserted to pMD18-T vector.The recombinant vector pMD18-T-ambp was identified by restriction endonucleases SalⅠ/ EcoRⅠand DNA sequencing.Results The ambp gene fragment was obtained by RT-PCR only from liver RNA,but not from kidney and pancreas.A DNA fragment of 1 098 bp,consistent with the size of ambp gene fragment,was generated by digesting the recombinant plasmid with the restriction enzymes SalⅠ/EcoRⅠ.The results of sequence analysis showed that the constructed AMBP cDNA sequence was identical to the published one in GenBank. Conclusion The human AMBP cDNA is obtained successfully and the recombinant cloning vector pMD18-T-ambp is constructed correctly.

To screen one kind of antitumor immunoadjuvant that could enhance the anti-tumor activity of heat shock fusion protein(HSP-MUC1).Methods MUC1-positive tumor bearing mice were subcutaneously injected by HSP-MUC1 combined with a novel CpG ODN or hIFNα2b,four groups were set up respectively as PBS,HSP-MUC1,CpG ODN(IFNα2b) and HSP-MUC1+CpG ODN(HSP-MUC1+IFNα2b).The tumor incidence and tumor volume in various groups were observed and calculated. Results The tumor incidence and tumor weight of mice in HSP-MUC1+CpG ODN group were lower than those in PBS group and HSP-MUC1 group(P<0.05); CpG ODN alone also displayed the similar anti-tumor effect as HSP-MUC1+CpG ODN,it could decrease the tumor incidence and tumor weight of mice compared with PBS and HSP-MUC1(P<0.05); And the tumor incidence and tumor weight of mice in HSP-MUC1+hIFNα2b group had no difference with those in PBS group and HSP-MUC1 group(P> 0.05). Conclusion CpG ODN could enhance the anti-tumor activity of HSP-MUC1 but need to be further determined for its optimal injection time；and human IFNα2b could not enhance the anti-tumor activity of HSP-MUC1.

To study the hypoglycemic activity of Injection of Ginseng Fruit Saponins (IGFS).Methods Fasting blood glucose (FBG),the contents of hepatic glycogen and the serum malondialdehyde (MDA) and activity of the serum super oxide dismutase (SOD) were determined in hyperglycemia rats induced by alloxan 14 d after administration of IGFS (in dosages of 7.5-30.0 mg•kg^-1 intraperitoneal injection).FBG and the content of hepatic glycogen were determined in hyperglycemia mice induced by adrenaline 7 d after administration of IGFS (in dosages of 10-40mg•kg^-1 intraperitoneal injection).At the same time,FBG and contents of the serum insulin and C-peptide were also observed,and insulin sensitive index (ISI) was calculated in normal rats pretreated with IGFS (in dosages of 7.5－30.0 mg•kg^-1 intraperitoneal injection). Results In hyperglycemia rats induced by 1.5% alloxan with sublingual vein injection,compared with normal group,FBG and content of the serum MDA in model group were increased significantly（P<0.05）,while the content of hepatic glycogen and activity of the serum SOD were decreased（P<0.05）; however,compared model group,FBG and content of the serum MDA were decreased significantly（P<0.01）,the content of hepatic glycogen and activity of the serum SOD were increased（P<0.05）by IGFS (in dosages of 7.5,15.0, and 30.0 mg•kg^-1 intraperitoneal injection for 14 d) in hyperglycemia mice induced by 0.1% adrenaline with subcutaneous injection.Compared with model group,FBG was reduced（P<0.01） and the content of hepatic glycogen was increased（P<0.05）by IGFS (in dosages of 20.0 and 　40.0 mg•kg^-1 intraperitoneal injection for 7 d).The content of the serum C-peptide was increased significantly（P<0.05 or P<0.01）while FBG and the content of serum insulin and ISI changed little in normal rats pretreated with IGFS (in dosages of 15.0 and 30.0 mg•kg^-1 intraperitoneal injection for 7 d). Conclusion IGFS possesses the hypoglycemic action on experimental hyperglycemia models induced by alloxan and adrenaline,which be related to scavenging the oxygen free radicals,restraining lipid peroxidation and increasing transformation and utilization of glucose.

To establish a multidrug resistance cell line from human prostatic cancinoma PC3M cell line.Methods The human prostatic cancinoma cell line PC3M was exposed to cisplatin with high dose and the concentration of cisplatin was increased gradually and then multidrug resistance cell line (PC3M/ CDDP) was established.The relative resistance was tested with MTT assay.The expressions of P-glycoprotein (P-gP) and multidrug resistance-related protein(MRP) were observed with immunocytochemical method.Results The IC₅₀ of cisplatin,mitomycin,doxorubicin,5-fluorouracil,vincristine on PC3M/CDDP cell were (0.603±0.102),(0.201±0.056),(0.267±0.067),(1.676±0.321) and (0.657±0.227) mg•L^-1,respectively,there was significant difference of IC₅₀ between PC3M/CDDP cell and PC3M cell(P<0.05).The MRP and P-gP expressions of PC3M/CDDP cell were higher than those of PC3M cells(P<0.05).The proliferation rate of PC3M/CDDP cell (40.5h) had no obvious difference when compared with that of PC3M cell (48.6 h) (P>0.05). Conclusion A multidrug resistance cell line (PC3M/CDDP) is established from human prostatic cancinoma PC3M cell line.The PC3M/CDDP cell was resistant to mulidrug,and no change on cell proliferation.It can be used to downstream experiment.

To study the mechanism of ginsenoside Rh₂ resistingRM-1 prostatic cancer cells in vitro. Methods After the RM-1 cells were treated with different doses of Rh₂,the cell viability was examined by MTT test.The techniques of morphology and flow cytometry were used to analyze apoptotic cycle of RM-1 cells and calculate apoptotic index.Results After RM-1 cells were treated with Rh₂,the growth of cell line was inhibited. When the doses of Rh₂ were 5,15,25, and 30 mg•L^-1,the apoptotic indexes were 12.0%,36.1%,51.2%， and 69.3%, respectively.The apoptotic rates were 41.9% and 48.9% when the doses of Rh₂ were 15 and 25 mg•L^-1.The RM-1 cells treated with Rh₂ showed a typical apoptotic appearance and hypodiploid peak before G₁ phase.Apoptotic index increased in time- and dose-dependent manner. Conclusion Ginsenoside Rh₂ can induce the apoptosis of RM-1 prostatic cancer cells and has significant anti-tumor effect.

To Study the antileukemic activity of donor-derived NK c ell in mice in vivo. Methods CB6F₁H-2b/d mouse model of EL9611 (H-2d) erythroleukemia was developed by injection of EL9611 (H-2d) cells in tail vein. 30 mice with erythroleukemia were randomly divided into 5 groups (six mice in each group). Experimental groups(2 groups): 2×10⁵ and 5×10⁵ NK cells of C₅₇BL/6H-2b were injected into mice after injected EL9611(H-2d) cells for 5 d in the first and the second experimental groups. Control groups（3 groups）: the first group was the one without treatment; the second one was the Ara-c-treated group which Ara-c were injected into mouse at 50 mg•kg^-1×6 d followed by infusion of EL9611(H-2d) for 5 d; the third one was syngeneic NK cell treated group, CB6F₁H-2b/d NK cells(1×106) were injected into CB6F₁H-2b/d mice after injected EL9611(H-2d) cells for 5 d. The effect was assessed by blood routine test, survival time, body weight, and histopathology in the recipients. Results ①There was no significant difference of life span between two experimental groups (P>0.05). The survival time of the second control group and experimental groups was much longer than that of the first control group (P<0.01); the survival time of the second of control group was much longer than that of experimental groups (P<0.01); the survival time of experimental groups was much longer than that of the third control group (P<0.01). Conclusion NK cell (H-2b) can delay erythroleukemia (EL9611, H-2d) CB6F1H-2b/d death time in erythroleukemia (EL9611, H-2d) CB6F₁H-2b/d model.

To investigate the contribution of transplantation of ex vivo expanded endothelial outgrowth cells （EOCs）from peripheral blood on repair of balloon injured vessels in rabbits. Methods Animals were divided into two groups(n=8): EOCs transplantation group and control group. The mononuclear cells（MNCs）were isolated from rabbit peripheral blood by density-gradient centrifugation and cultivated in EGM-2, yielding EOCs. A rabbit model of balloon catotid injury was used to evaluate the effects of EOCs transplantation on endothelial regeneration and inhibition of neointimal formation. Immediately after denudation, EOCs（the second passage）in 100 μL saline or 100 μL saline alone (control) were administered into the lumen of injured artery. Meanwhile the cells were labeled by CM-DiI for cells tracking. Four weeks after transplantation, rabbits were killed. The endothelial regeneration rate (RA/TA) and IA/MA ratio in fluorescence-labeled EOCs were detected. Results Four weeks after transplantation, fluorescence-labeled cells were found in the media, neointima and on the luminal surface of injured segments. Evans blue staining result demonstrated that RA/TA ratio in EOCs transplantation group (89.1%±6.3％) was much higher than that in control group (62.1%±7.5%, P<0.01). Meanwhile, compared with control group, there was a significant decrease of neointimal formation in EOCs transplantation group. The IA/MA ratios were 0.88±0.14 and 0.44±0.06, respectively(P<0.01）. Conclusion Transplantation of ex vivo expanded EOCs to balloon-injured arteries can accelerate reendothelialization significantly and reduce neointimal formation.

To observe the effects of Okinawa Habu apoxin protein-1 (OHAP-1) on the proliferation inhibition of rat C6 glioma cells and its mechanisms. Methods MTT colorimetric analysis was used to measure the inhibitory effect of OHAP-1 with different doses（2.5,5.0,and 10.0 mg•L-1） on C6 glioma cells .RT-PCR was used to evaluate the mRNA expressions of bcl-2 and bax genes.The activity of superoxide dismutase (SOD) and the level of maleicdialdehyde (MDA) in the C6 glioma cells were also examined. Results The proliferation of C6 glioma cells was significantly inhibited by different doses of OHAP-1（2.5,5.0,and 10.0 mg•L^-1）.The inhibitory rate were 49.77%,67.65％,and 76.42%，respectively. The inhibitory rate in 2.5,5.0, and 10.0 mg•L^-1 groups were higher than that in control group(P<0.001).With the increasing of the dose of OHAP-1,bax mRNA expression increased,and bcl-2 mRNA decreased markedly,the ratio of bcl-2/bax mRNA also decreased.In 2.5,5.0, and 10.0 mg•L^-1 OHAP-1 groups,MDA concentrations increased significantly compared with control group(P<0.01),and SOD decreased (P<0.01); In 5.0 and 10.0 mg•L^-1 OHAP-1 groups,MDA concentrations were higher than those in 2.5 mg•L^-1 OHAP-1 and control groups (P<0.001);in 10.0 mg•L-1 OHAP-1 group, SOD activity was lower than those in 2.5 mg•L^-1 and control groups (P<0.001); in 10.0 mg•L^-1 OHAP-1 group, SOD activity was lower than those in 5.0 ,2.5 mg•L^-1OHAP-1 and control groups (P<0.001). Conclusion The proliferation inhibition of C6 glioma cell induced by OHAP-1 may be related to the expression of bcl-2/bax mRNA decreasing resulted from oxidative stress.

To investigate the expressions of nitric oxide (NO) and plasminogen activator inhibitor type-1（PAI-1）in rabbit models of atherosclerosis and the interference effect of atorvastatin. Methods Twenty four male Japanese rabbits were divided into 3 groups (8 in each group): high-fat diet group（group A）,high-fat plus atorvastatin group (group B) and normal diet group（group C）.The expressions of NO and PAI-1 were detected with ELISA assay.Plasma lipid was detected with automatic analysis divice. Results The espression of NO in group B was significantly higher than those in groups A and group C (P<0.01); there was no significant difference between groups A and C (P>0.05).The expression of PAI-1 in group B was significantly lower than those in groups A and C,there was no significant difference between groups A and group C (P>0.05).The levels of serum TC in groups A,B,and C were （23.51±10.58）,（14.27±3.51）and（1.36±0.33）mmol•L^-1, respectively; the levels of serum LDL in groups A,B,and C were （21.39±10.00），（14.23±4.01）and（0.72±0.35）mmol•L^-1;there were significant differences between three groups (P<0.05),while TG had no significant difference between three groups (P>0.05). Conclusion High-fat diet has no significant effect on the expressions of NO and PAI-1 in atherosclerosis rabbits .Atorvastatin can increase the expression of NO and decrease the expression of PAI-1,and can inhibit the progression of atherosclerosis.

To study the effects of LaCl₃ on P16 and P21 expressions of hepatocellular carcinoma cells. Methods The experiment was divided into 4 groups.In experiment groups,CBRH-7919 cells were cultivated with 0.01,0.10 and 1.00 mmol•L-1 LaCl₃ in DMEM containing 10％ calf serum.In control group,CBRH-7919 was cultivated in DMEM containing 10％ calf serum without LaCl₃.The growth of CBRH-7919 cells was observed following treated with 0.01,0.10,1.00 mmol•L^-1 LaCl₃ for 1,3,5 d in vitro, respectively,the changes of cell cycle were detected by flow cytometry.At the same time,P16 and P21 were detected by immunocytochemical analysis. Results Compared with control group,the inhibitory rate of growth of CBRH-7919 cells was increased evidently when treated with 0.1 and 1.00 mmol•L^-1 LaCl₃ for 3 and 5 d（P< 0.01）,the percentage of CBRH-7919 cells ofG₀/G₁ was evidently increased（P< 0.01）,the expressions of P16 and P21 were increased（P< 0.01）. Conclusion LaCl₃ can obviously inhibit the growth of CBRH-7919 cells by up-regulating P16 and P21 that can block the cell cycle progress from G₁ to S.

To observe the effects of Compound Acanthopanax Senticosus Injection (CASI) on experimental arrhythmia induced with medicine in rats and cobayaes. Methods The experimental arrhythmia models were induced with barium chloride (BaCl₂),uabaina and aconitine in rats and cobayaes.The incidence rate and mortality rate of arrhythmia，the emergence time and the duration of arrhythmia induced with BaCl₂ were recorded in rats pretreated with CASI (in dosages of 25,50, and 100 mg•kg-1 sublingual vein injection).At the same time,the dosages of uabaina and aconitine were observed when the ventricular premature contraction (VP),ventricular tachycardia (VT),ventricular fibrillation (VF) and cardiac asystole(CA) were appeard in rats and cobayaes pretreated with CASI (in dosages of 25,50, and 100 mg•kg^-1 sublingual vein injection). Results Compared with control group,the incidence rate of arrhythmia induced with BaCl₂ was lowered (P<0.05),the emergence time of arrhythmia was delayed(P<0.01),the duration of arrhythmia was shortened(P<0.05),and mortality rate of arrhythmia was decreased significantly (P<0.01) in rats pretreated with CASI in dosages of 25,50, and 100 mg•kg^-1sublingual vein injection.The tolerance doses of cobayaes on VP,VT,VF and CA induced with uabaina were significantly higher in 50 and 100 mg•kg^-1 CASI groups than control group (P<0.05 or P<0.01), while the tolerance doses of rats on VP,VT and VF induced with aconitine were also significantly higher in 100 mg•kg^-1 CASI group than control group (P<0.05). Conclusion CASI have a significantly protective effect on experimental arrhythmia induced with medicine in rats and cobayaes,its effect on calcium channel is more better than on natrium channel.The results show that CASI may be calcium channel blocker.

To investigate the effects of valsartan on experimental left cardiac function and the vasoactive substance in rats with acute myocardial infarction (AMI). Methods Seventy Wistar rats were randomly divided into 6 group:①Sham groups: including Sham 1(n=5) and Sham 4(n=5).The pericardium of rats in Sham groups were cut open and sutured immediately.The rats were routinely breeded for 1 week and 4 weeks,1.5 mL saline was poured into stomach once a day.②Control groups: AMI 1 (n=10) and AMI 4 (n=10).Anterior descending branches of coronary artery of Wistar rats were ligated to establish models of AMI.The rats with AMI were poured into stomach with 1.5 mL saline once a day after AMI for 1 week and 4 weeks.③Valsartan groups: VAS 1 (n=10) and VAS 4 (n=10).The rats with AMI were poured into stomach with valsartan 10 mg•kg^-1 and 1.5 mL saline for 1 week and 4 weeks(once a day).Cardiac function was assayed by arterial cannulatio.Angiotensin Ⅱ（Ang Ⅱ）,aldosterone (ALD),endothelin (ET),thromboxane A₂(TXA₂) and prostacyclin I₂ (PGI₂) in serum were measured by radioimmunoassay.Results ① Valsartan could evidently improve cardiac function on the 1st and 4th week after experimental AMI.+dp/dt_max and -dp/dt_max were both increased on the 1st and 4th week and more evidently on the 4 week.②Compared with control groups,valsartan decreased the levels of ALD,ET and TXA₂ in plasma and increased the levels of AngⅡ and PGI₂ in plasma. Conclusion Valsartan could improve left cardiac function on late stage of infarction,the effect improve not only systolic function,but also diastolic function.

To investigate the protective effect of heme oxygenase-1 (HO-1) induced by doxorubicin on hepatic ischemia-reperfusion in rats and its mechanism. Methods Fourty eight male Wistar rats were divided randomly into six groups (eight rats in each group)：normal group(N)，doxorubicin group(DOX)，ZnPP group，ischemia reperfusion group(IR)，DOX +IR group(DOX-IR) and DOX +ZnPP+IR group.The contents of serum alanine aminotransferase(ALT)，aspartate aminotransferase(AST) and lactate dehydrogenase(LDH) were examined as liver function indicators.Serum endothelin（ET）and tumor necrosis factor-α（TNF-α） were also measured as markers of stress.HO-1 protein expression was measured respectively by Western blotting and immunohistochemical method (S-P).Ultrastructural changes were investigated by transmission electron microscope. Results The values of ALT，AST，ET-1, and TNF-α in DOX -IR group were significantly lower than those in IR group (P<0.05)，but there was no significant difference between DOX group and normal group (P>0.05).There was no significant difference in liver function test between DOX -ZnPP-IR group and IR group (P>0.05).The results of Western blotting and immunohistochemical method showed that HO-1 protein expression increased in DOX group and DOX -IR group，and was negative in normal group.More distruction of ultrastructural changes of hepatic cells in IR group was found than in DOX-IR group by transmission electron microscope. Conclusion Doxorubicin pretreatment could protect the liver from ischemia-reperfusion injury，which may be related to HO-1 expression induced by doxorubicin,low dosage of doxorubicin does little harm to rat liver.

Abstract:Objective To investigate the effect of pyrrolidine dithiocarbamate (PDTC) on the proliferation and expression of IL-8 of SKOV3 cells in acid environment,and the role of nuclear factor κB (NF -κB) in the process.Methods SKOV3 cell lines were divided into 5 groups: normal culture group,acid environment group，acid environment plus PDTC groups (25,50 and 100 mol•L^-1 PDTC).All the SKOV3 cells were cultivated for 12 h.The effect of PDTC on the proliferation of SKOV3 in the acid environment was determined by MTT,the content of IL-8 protein in the culture supernatant was detected by ELISA and the expression of NF-κB protein was measured by Western blotting.Results The IL-8 expression level in the acid environment was(1 384.211±42.320) ng•L^-1.there was significant difference compared with normal group（617.505 ng•L^-1±47.850 ng•L^-1)(P< 0.01).The IL-8 expression levels were decreased gradually with the enhancement of PDTC concentration (25,50, and 100 mol•L^-1),and the values were （1 239.053±14.490）,（1 113.578±29.140）and（1 014.457±55.49０） ng•L^-1，there were significant differences compared with acid environment group (P<0.05)．The deeper color protein strap of NF-κB in the SKOV3 cells was found in the acid environment and the color turned pale after SKOV3 cells were treated with PDTC. Conclusion PDTC could suppress the secretion of IL-8 in acid environment by supressing the activation of NF-κB.

Abstract:Objective To investigate the effect of silicone oil on the corneal endothelium in experimental animal models,while the silicone oil in the anterior chamber of the experimental animals will be taken out so as to explore their corneal changes.Methods Forty healthy white rabbits were randomly divided into three groups : silicone oil in the anterior chamber group (SIG),silicone oil in and out of the anterior chamber group (SIOG) and control group(CG).The cornea of rabbit was investigated at different times by slit-lamp,corneal thickness instrument,endothelial cell count instrument,histology and electromicroscopy.The time of silicone oil in the anterior chamber without keratpathy and the pathological changes of cornea with taking out of the silicone oil were observed.Results The density of the corneal endothelium in SIOG and SIG decreased more obviously by endothelial cell count instrument from 8 weeks to 16 weeks compared with control group (P<0.01).The results of histology and electron microscopy showed that the core of cells shrank,many layers of corneal endothelium had been found.Cornea of SIOG was edematous by the slit-lamp.Compared with SIG,the corneal thickness in SIDG increased significantly (P<0.01);the fibers of corneal stroma in SIOG were loose and disorder.The corneal stroma thickness in SIOG increased lightly(P<0.01).The cornea in CG didn’t change obviously.Conclusion The damage of corneal endothelium of rabbit eyes will not recover if the silicone oil is in the anterior chamber beyond 8 weeks,more time come with more damage.

Abstract:Objective Through pathological and histological examination on retina to research the dosage relevance of trace PFD with toxicity in rabbit retina,and provide experimental reference basis for clinical practice.Methods Fourty eight eyes from 24 experimental rabbits were randomly divided into experimental group(36 eyes) and control group(12 eyes).The experimental one was divided into 3 groups,12 eyes in each group.The surface of rabbit retina was directly injected with 30,50,and 100 μL liquids of PFD and BSS.The conditions of rabbit retina were observed under ophthalmoscope every day after the liquid injection.The sections of retina tissue were observed under transmission electron microscope in each experimental and control groups at the 4th,7th,14th and 21th day after injection.Results All samples were normal by ophthalmoscope examination while no pathological change of retina was found at various time points .At the 4th day,none substantial damage had been made in any dosage groups by transmission electron microscope examination except for 100 μL group.Only one retina of 100 μL group showed that the space around cell nucleus broadened appreciably,slightly atactic nucleus and slightly compact cytoplasm.It showed some early changes.From the 7th day,similar pathological changes were found: damage on outer segments of light receptor and outer plexiform layer,properties changes of light receptor，forming of vesicles of inner nuclear cells,damage of ganglion cell,dying of inside retina,actions of gulping cells in retina,swallow of film plates by pigment epithelial cell of retina,and dropping of top fine hair. But the retina of control group had no pathological changes.Conclusion Trace PFD exists in eyes.There is no relevance between poisonous development of retina and remaining dosage of PFD.However,large remaining of PFD can lead to retina toxicity early.

Abstract:Objective To study the effect of Shuganjieyuyin (SGJYY) on monoamine transmitters and their metabolites of brain tissue in depressive mice.Methods The lonely feeding and chronic moderate intensive unpredictable stimulation were used to build depression model and SGJYY was administered. HPLC was used to test the contents of monoamine transmitters and their metabolites of brain tissue in depressive mice and observe the changes of weight and consumption of sugar water and behaivor indexes in mice.Results Compared with normal group,the weight increased slowly(P<0.01), the score of Open-field and sugar water consumption decreased(P<0.01)，the time of no-moving increased(P<0.01) in model group.Compared with normal group,the contents of 5-HT,NE,DA and DOPAC of brain tissue decreased(P<0.01),5-HIAA and MHPG increased(P<0.01) in model group.The 5-HT contents in BYJ and large dosage SGJYY groups were near normal level,the 5-HIAA content in middle dosage SGJYY group was near normal level.The contents of NE in BYY and large dosage SGJYY groups were near normal level,the contents of MHPG in BYJ and anti-depression of SGJYY groups were near normal level.The contents of DA in BYJ and large SGJYY group were near normal level,the DOPAC content in BYJ group was near normal level.Conclusion The mechanism of anti-depression of SGJYY is related to regulating the contents of monoamine transmitters and their metabolites.

Abstract:Objective To observe the changes of transforming growth factor β1 (TGF-β1) and its receptor(TGF-βRⅠ) in the uterus in androgen-induced sterile rats(ASR).Methods Twenty two SD female rats of 9-day age were randomly divided into two groups:ASR model group(n=12) and control group(n=10).ASR model was established by injecting testoserone propionate into rats subcutaneously. At the same time,the rats in control group were injected with neutral tea oil with same volume.The level of serum testosterone(T) was determined by means of radioimmunoassay.The expressions of TGF-β1 and TGF-βRⅠin the uterus were detected by immunohistochemistry method.The morphologic changes in the uterus by HE staining were observed. Results Compared with control group,the level of serum testosterone in medol group was increased obviously(P<0.01),the expressions of TGF-β1 in the endometrium and myometrium were decreased significantly(P<0.01), the expression of TGF-βRⅠ in myometrium was decreased obviously(P<0.05),while the expression of TGF-βRⅠin endometrium was increased significantly(P<0.05). In model group ,myometrium of the uterus was tenuis,endometrium was atrophic and the number of glandular organs was decreased.Conclusion The expressions of TGF-β1/ TGF-βRⅠin endometria and myometrium in ASR are abnormal,the differentiation of endonetrium and the growth of myometrium are injuied.These changes might be one of the reasons to lead to infertility.

Abstract:Objective To investigate the expression of c-jun in precancerous lesion and squamous cell carcinoma tissues of hamster buckle pouch mucosa.Methods The models of premalignant and malignant lesions were established in hamster buckle pouch mucosa induced with DMBA.The expressions of c-jun in 23 oral normal mucosa, abnormal epithelial hyperplasia and 29 oral squamous cell carcinoma (OSCC) were measured with immunohistochemical SABC method.Results c-jun mainly expressed in basal cells and prickle cells in normal mucosa,simple and abnormal epithelial hyperplasia,its positive expression was in nucleus.c-jun expressed mainly in various layer cells of OSCC,its positive expression was in nucleus and cytoplasm.There were significant differences of positive expression rate between various groups(χ²=13.71，P<0.01).The positive expression rates in abnormal epithelial hyperplasia(77.2%) and OSCC(55.17%)were higher than that in normal mucosa(26.09%)(P<0.05).The positive expreesion rate in abnomal epithelral hyperlasia was higher than that in simple epithelial hyperlasina(36.36%)(P<0.05). Conclusion c-jun may play an important role in malignant transformation of oral benign lesions.

Abstract:Objective To examine the effects of formaldehyde on the process of sperm development and reproductive capacity in male mice and the micronucleus rate of liver cells from offspring.Methods The mice were randomly divided into negative control group（NC）,formaldehyde exposed group and positive control group (PC).The exposed dose was 21 mg•m-3 (1/24 LC₅₀),42 mg•m-3 (1/12 LC₅₀) and 84 mg•m-3 (1/6 LC₅₀),respectively. 12 male mice were included in each group.Male mice from different formaldehyde exposed groups were inhaled with formaldehyde for 2 h one day,6 d each week,lasted for 13 weeks.The aberration rate of sperm in male mice and the micronucleus rate of liver cells from offspring were observed,and the reproductive capacity of male mice was detected by dominant lethal test.Results The aberration rates of sperm in mice from different formaldehyde exposed groups were significantly higher than that in negative control (P<0.05),and the relationship between the spermatozoa aberration rate and the concentration of inhaled formaldehyde was positive correlation (r=0.83,P<0.001).The rates of fetal absorption in pregnant mice from three different exposed groups were significantly higher than that in negative control (P<0.01),and the number of live offspring from 1/6　LC₅₀ group was lower than that in negative control (P<0.05).Conclusion Formaldehyde can cause the increasing of sperm aberration rate and the decreasing of the reproductive capacity in male mice.The results suggest that formaldehyde can affect the genetics of male mice.

Abstract:Objective To explore the possibility of reconstruction of tissue engineering artificial skin with modified poly lactic acid (PLA).Methods Fibroblasts(FB) and keratinocytes(KC) from new born rats isolated by treatment of Dispase-trypsin and collaganase were cultivated.Tissue engineering artificial skin was reconstructed by planting subcultured FB（3.0×10⁵cell•mL^-1）and KC（5.0×10⁵cell•mL^-1）separately onto the modified PLA,which was made by special ways and it’〖KG-3〗s mechanism function was improved,and cultivated for one week. Results The artificial skin was composed of epidermis and dermis and similar with natural skin.The skin consisted of FB and KC was at various proliferation and differentiation stages.FB and KC increased on the surface of PLA. Conclusion Dialdehyde starch(DAS) not only improves the mechanism function of PLA,but also has good effect on cells.And it doesn’〖KG-3〗t affect the growth and metabolism of cells.So it is feasible as scaffold of tissue engineering artificial skin.

Abstract:Objective To investigate the association between serum phospholipase A2 (PLA2) activity and schizophrenia in Han population in North China.Methods The acid-base titration method was conducted to examine the serum PLA2 activity in 230 subjects including 115 patients with schizophrenia (case group) and 115 matched healthy controls(control group).Results The serum PLA2 activity level in case group was significant higher than that in controls（P<0.001）.In subjects aged 10 to 20 and above 60 years,there were no significant differences of serum PLA2 activity between cases and controls（P>0.05）; in other subjects aged 21 to 30,31 to 40,41 to 50,51 to 60 years,there were significant differences in distribution of serum PLA2 activity between cases and controls（P<0.05）.Conclusion The augmentation of serum PLA2 activity may be associated with the onset of schizophrenia.

Abstract:Objective To establish the method for isolation and cultivation of mesenchymal stem cells(MSCs) from human embryo in vitro,and to identify their biological characteristics. Methods Cells were separated from human embryo 5-6 weeks after digestion with 0.25% trypsin for 7-10 min,and the anchoring cells were expanded in MSCs media.Flow cytometry(FCM) was adopted to determine the immunophenotypes of MSCs,and the proliferation ability and cell cycle were etected by MTT and propidium iodide(PI) staining. Different induction conditions were used to identify the multipotent differentiation.Results MSCs were obtained from human embryo by tissue digestion. FCM result showed the immunophenotype of MSCs was positive for CD29,CD44,CD73,CD105 and CD166,and negative for CD34,CD45 and HLA-DR.The doubling time of MSCs was 23 h.Lipid droplets were found in lipo-induced MSCs，and under ossa-induced condition,the histological analysis of the cells revealed stronger expression of alkatnephosphatase and the mineralization of calcium salts was confirmed by alizarin red . Conclusion The phenotype of MSCs derived from human early embryo is coincident with adult MSCs,and these cells have the potential to differentiate into adipocytes and osteoblasts.

Abstract:Objective To investigate the relationship between two excision repair cross-complementation group 1(ERCC1) polymorphisms, 8092C>A and 19007G>A, and susceptibility to acute lymphoblastic leukemia (ALL) in children . Methods A case-control study consisting of 183 children ALL patients and 190 controls in a Chinese population was performed to examine the ERCC1 genotype. Results For the ERCC1 8092C> A polymorphism, individuals carrying the CC genotype had a significant high risk when compared with carrying one A allele gene (AA /AC) patients (P = 0.030). For the ERCC1 19007G >A polymorphism, no association was found between ERCC1 polymorphism and ALL risk. Stratification for sex with regard the ERCC1 8092C > A and 19007G >A polymorphism form showed a highly significant risk (OR=1.94, 95% CI 1.09-3.41, P=0.020;OR=2.36, 95%CI 1.05-5.27, P=0.037) of ALL among males whereas that of females did not show any significant risk. For the ERCC1 8092C> A polymorphism, individuals under 10 years old carrying CC genotype had significantly risk (OR=1.76，95% CI 1.04-2.99; P=0.04). However, the ERCC1 19007G >A polymorphism was not significantly associated with ALL in age. Conclusion These results suggest that the ERCC1 8092CC gene polymorphism is related to the occurrence of ALL in Chinese children.

Abstract:Objective To study the change of chromosome aberration rate and micronucleus rate in patients with esophageal carcinoma before and after radiotherapy.Methods Twenty one patients with esophageal carcinoma who were fit for conditions were divided into two groups randomly.GroupⅠ（n=10）,patients received conventional radiotherapy（radiation field was regular rectangular shape）；groupⅡ(n=11),patients received conformal radiotherapy（radiation field was same to the shape of target volume）,1.8-2.0 Gy,one time a day,5 times a week,the total dose was 60-70 Gy.The peripheral blood was obtained before and after radiotherapy,chromosome aberration rate and micronucleus rate were detected.Results The chromosome aberration rate and micronucleus rate were increased after conventional radiotherapy than before （P<0.05），they were also increased after conformal radiotherapy than before,but there was no statistics difference（P>0.05）.Conclusion Compared with the conventional radiotherapy,the conformal radiotherapy can decrease cytogenetics damage,the detection of chromosome aberration rate and micronucleus rate can be regarded as a simple method to evaluate cytogenetics damage in esophageal carcinoma patients induced by radiotherapy.

Abstract:Objective To study the change of matrix metalloproteinase-2 (MMP-2) in the development of renal fibrosis and evaluate the diagnostic value of blood MMP-2 level in renal fibrosis.Methods Thirty three patients with chronic glomerulonephritis were divided into proliferation group(n=18) and fibrosis group(n=15) according to renal fibrosis degree in renal pathology.Another 8 healthy subjects were used as control group.The levels of pro-MMP-2 and MMP-2 in serum were measured with the method of SDS-PAGE enzymography.Then the levels of pro-MMP-2 and MMP-2 in serum were compared between various groups.Results The serum MMP-2 level in proliferation group was much higher than that in control group(P<0.01) and it was lower in fibrosis group than that in control group(P<0.05).Compared with proliferation group,the content of urine protein in fibrosis group decreased obviously(P<0.01),serum fibrinogen level increased markedly(P<0.05),FN increased (P<0.01);there were no obvious differences of plasma lipid and creatinine level(P>0.05).Conclusion There is a significant difference of serum MMP-2 between suffering group and normal control group.Therefore the detection of serum MMP-2 level can be conducive to understand the development of disease and estimate prognosis.

Abstract:Objective To evaluate the effect of Aiyishu injection on the cellular immunity state of body and toxic and side reaction in post-operation patients with breast cancer after chemotherapy Methods Seventy patients with breast carcinoma at stage Ⅰ-ⅢA were randomly divided into treatment group (Aiyishu associated CAF regimen) (n=36) and control group (CAF regimen) (n=34)The activities of T cells and NK cells were examined in two groups before and after treatment,and the toxic, side reaction and living quality were also comparedResults The levels of CD3,CD4,CD8 and the ratio of CD4/CD8 and the activity of NK cells in treatment group were higher than those in control group (P<0.05);the number of aleucocytosis patients in treatment group was lower than that in control group(χ²=7.44,P=0.006) In treatment group,KPS increased in 36.11%(13/36) patients,and it was higher than that in control group (11.76%，4/34)(χ²=5.64,P=0.018);44.44% patients put on weight,while 17.65% in control group(χ²=6.66,P=0.036)Conclusion Aiyishu injection can increase the ability of immunity of body and reduce the toxic and side reaction,and improre the living quality in patients with breast cancer when it is used in post operative chemotherapy of breast cancer.

Abstract:Objective To investigate the influence of sufentanial in postoperative patient controlled epidural analgesia(PCEA) in hemorheology after total hip replacement and its inhibitory effect on thrombosis.Methods Fifty patients,ASA Ⅰ-Ⅱ,undergone total hip replacement were randomly divided into PCEA group (n=25) and control group (n=25), and received continuous epidural anesthesia.After operation,5 mL 0.2% ropivacaine was administered in PCEA group,and then PCEA pump was used,analgesia liquid included 0.4 mg•L^-1 sufentanial,0.2% ropivacaine and saline.Petidine was administered intramuscuarly according to pain in control group.VAS scores 1,12,24 and 48 h after operation and changes of various parameters of hemorheology at diffenent time after anesthesia were observed.Results ①The VAS scores 1,12,24,48 h after operation were lower than those in control group (P<0.05).②The plasma viscolities and fibrinogen levels in two groups 1 h after anesthesia and at the end of operation were significantly lower than before anesthesia (P<0.05);48 h after operation,they were higher in control group than those in PCEA group(P<0.05).The viscosity shear rates of whole blood in two groups 1 h after anesthesia and at the end of operation were lower than before anesthesia(P<0.05). Conclusion Sufentanial in PCEA can improve the hemorheological parameters after total hip replacement and can be used as a effective method to reduce thrombosis.