Abstract:ObjectiveTo explore the effect of high-mobility group mucleosome binding domain 5(HMGN5) by RNAi on proliferation and cell cycle of lung cancer cell line H1299，and provide a theoretical basis for lung cancer targeted therapy. Methods SiRNA targeting HMGN5 gene was cloned into lentivirus vector.SiRNA-HMGN5 lentivirus particles were infected into H1299 cells in order to silence the expression of HMGN5. Negative control group and RNAi group were set up. The interfering efficiencies of the plasmids on HMGN5 gene were detected at the mRNA and protein levels by Real-time PCR and Western blotting. The proliferation  of H1299 was analyzed by MTT and BrdU assay. The cell cycle was detected by flow cytometry. Results Compared with negative control group,the expression of HMGN5 mRNA in RNAi group was down-regulated by 50.7%(P< 0.05);the protein level was significantly deceased(P<0.05). Compared
with negative control group,the proliferation rate of H1299 cells was deceased by MTT assay. BrdU assay results showed that the cell proliferation rate of  H1299 in  RNAi group (37.8%) was significantly lower than that in negative control group(55.0%)(P< 0.05).The percentage of cells at G1 phase（54.6%±0.9%） in  RNAi group was inceased compared with negative control group（46.5%±0.4%）(P< 0.05). Conclusion Silencing HMGN5 gene can inhibit the proliferation of H1299 cells.It shows that HMGN5 plays a role in the development of lung cancer and it may be beneficial in finding a new therapy for lung cancer.

Abstract:Objective To detect the expressions of Ets family transcription factors Ets1 and Ets2 in mouse testis tissue  and explore the effects of Ets1 and Ets2  on the development of mouse testis  and  self-renewal and differentiation of spermatogonial stem cells(SSCs). Methods The mouse testis tissues were collected from specific developmental stages including postnatal days 1,5,10,15,20,25,30,35,40,50 and 70;Busulfan peritoneal injection was performed and mouse testis tissues were collected on the 0th,3rd,5th,8th,10th,18th days after injection, respectively. The mRNA expression levels of Ets1 and Ets2 in samples were analyzed by semi-quantitative RT-PCR with β-actin as the internal control. Results The expression of Ets1 was significantly higher during the period of postnatal 1-30 d than that at postnatal  day 35 (P<0.05 or P<0.01),while its expression was decreased evidently and maintained at a stable level afterwards. The expression of Ets2 was  significantly higher during the period of postnatal 1-25 d than that at postnatal day 35 (P<0.05 or P<0.01),while it was decreased significantly and maintained at a stable level afterwards similarly. After busulfan treatment,the expression of Ets1 was declined and reached the lowest level at day 5,then was increased gradually and reached the level of day 0 after busulfan treatment and maintained steadily around day 9. Notably,the expressions of Ets1 at day 5 and day 8 were significantly higher than that of day 0 after busulfan treatment (P<0.05 or P<0.01). No obvious changes were observed for the expression of Ets2 during 1-9 d after busulfan treatment,while it was decreased dramatically at day 10,which was significantly lower than those of day 0 and day 18 (P<0.05 or P<0.01). The expression of Ets2 was gradually increased after day 10 and reached its normal level around day 18. Conclusion Ets1 and Ets2 may affect the early development of mouse testis,adult spermatogenesis,as well as the proliferation and differentiation of SSCs.

Abstract:Objective To observe the effects of oleanolic acid (OA) on apoptosis and the expression of Bcl-2 protein in HL-60 cells of the human leukemia implant model mice and investigate the therapeutic effect mechanism of OA on the human leukemia implant model mice. Methods 16 SCID mice were injected with 0.5 mL HL-60 cells(2×10⁷mL-1,cultivated in vitro),then the human promyelocytic leukemia implant models of mice were estabished.The model mice were divided into model group and treatment group(the mice were treated by subcutaneous injection with  200 mg.kg^-1 OA;at the same time,8 normal SCID mice were used as control group.2 weeks after treatment,the general condition and WBC classification in peripheral blood and bone marrow were observed.The invasive depth of HL-60 cells in spleen was tested by histopathologic examination;the apoptotic rate of HL-60 cells in spleen invasive leukemic was measured by TUNEL;the expression of  apoptosis related gene Bcl-2 was also detected by immunohistochemistry. Results The HL-60 cell implant models of SCID mice were established successfully;the bodyweight of the mice in treatment group(15.0 g±0.8 g) was obviously higher than that in control group(13.9 g±0.9　g) (P<0.01).The survival time of the mice in treatment group(50.3 d±5.5 d) was much longer than that in control group(37.1 d±4.4 d)(P<0.01).Compared with model group,the HL-60 cells in peripheral blood in treatment group trended to differentiate into normal WBC(P<0.05),the number of erythroid cells was reduced in bone marrow(P<0.05),the invasion of HL-60 cells in spleen was notably reduced(P<0.05),the apoptotic rate of HL-60 cells was increased(P<0.01) and the expression of Bcl-2 was decreased(P<0.01). Conclusion The HL-60 cell implant models of SCID mice are established successfully;OA could improve the general condition and increase the survival time of the leukemia implant model mice.OA could induce the apoptosis of HL-60 cells through down-regulation of Bcl-2 expression.

Abstract:Objective To explore the possibility to damage mice pancreas by recombinant fusion protein vaccine,heat shock protein 65-mucin 1 (HSP65-MUC1) via molecular mimicry.Methods 21 C57BL/6 mice were randomly divided into three groups:PBS control,HSP65 and HSP65-MUC1 groups.The mice in each group were injected underneath with PBS,HSP65 and HSP65-MUC1,  respectively,once a week for three weeks. The mouse  pancreas were isolated under aseptic situation,the proliferation of specific lymphocytes was   tested by flow cytometry,the pathological changes of mouse pancreas were detected with histo-pathological analysis. Results By flow cytometry,compared with   PBS and HSP65 groups,the lymphocytes specific to HSP65-MUC1 of mice in HSP65-MUC1 group were increased by 16.88%（P<0.001）,the lymphocytes specific to HSP65 were increased by 7.29 %（P<0.01）;the lymphocytes specific to HSP60 were increased by 5.79 %（P<0.05). However,HE staining of mouse pancreas in immunized mice kept normal. Conclusion The mouse pancreas are not injured by HSP65-MUC1 via molecular mimicry.

Abstract:Objective
To study the influence of aprotinin in hepatocyte proliferation ability in rats with experimental chronic liver injury and provide thoreotical basis for study on protective effect of aprotinin on chronic liver injury.  Methods Wistar rats were randomly divided into normal control group,carbon tetrachloride (CCl4) model group,low dose  aprotinin group,midde dose aprotinin group,high dose aprotinin group and hepatocyte group.Immunohistochemical method was used to observe the proliferating cell nuclear antigen (PCNA)-positive cell number in various groups.  Results The positive cell number in control group was (4±2)/vision;(5±2)/vision in model group;(39±13)/vision,(49±14)/vision and (57±12)/vision in low,middle,and high dose groups;(26±8)/vision in hepatocyte group. The positive cell numbers in aprotinin groups were markedly increased compared with model group（P<0.05）,the positive cell number was increased with the increase of aprotinin dose. The positive cell numbers in aprotinin groups were more than that in hepatocyte group（P<0.05）.The proliferation of PCNA positive cells in each aprotinin group was significantly increased,especially in high dose aprotinin group. Conclusion The aprotinin could promote the proliferation of chronicly injured liver cells and this effect could be enhanced by the increasing of aprotinin dose.

Abstract:Objective
To evaluate the effects and mechanisms of taurolidine in combination with X-rays irradiation on cell cycle progress of murine melanoma cells,and  provide a new idea of malignant tumor clinical treatment. Methods B16-4A and B16-F10 cells were divided into 4 groups according to the  concentrations of taurolidine (0,25,50 and 100 μmol.L^-1 ),respectively and combined with 1,2 and 4 Gy X-rays irradiation. The apoptotic  rate and cell cycles were detected by flow cytometry. The expressions of cyclinB,cdc2,and caspase-3 were detected by Western blotting. Results  Compared with 25  μmol.L^-1  taurolidine  group,the arrest of G2/M phase of B16-4A and B16-F10 cells after treated with 50 and 100  μmol.L^-1 taurolidine was found and the cell numbers  were increased by 54.9%,73.7% and 36.8%，55.5%,respectively(P<0.05).The arrest of G2/M phase of B16-4A and B16-F10 cells was also found and the cell numbers were decreased by 52.1%，44.2% and 59.3%，52.7%(P<0.05),respectively, after treated with  50  μmol.L^-1  taurolidine in  combination with 2 and 4 Gy X-rays irradiation. Therefore,the increase of caspase 3 expression  and the decrease of cyclin B and cdc2 expressions were induced by the combination of 50  μmol.L^-1  taurolidine with 2 and 4 Gy X-rays irradiation  compared with control,taurolidine alone and irradiation alone groups.Conclusion Taurolidine in combination with X-rays irradiation can delete the _G2/M arrest,supress the expressions of cyclin B and cdc2,increase the expression of caspase-3 and induce the apoptosis.

Abstract:Objective
To study the effects of 20(s)-proto-panaxdiol（PPD）on transcription and protein expressions of caspase-9 and caspase-3,and content of cleaved caspase-3 in Siha cells in vitro,and clarify the mechanism of its apootosis action. Methods Siha cells cultivated in vitro were treated with alcohol (negative control group)and 20 μg?L-1 PPD (20  μg.L^-1 PPD group),respectively. The apoptosis was detected by FCM 48 h after treatment. The transcription and protein expressions of caspase-9 and caspase-3 in siha cells were analyzed by RT-PCR,Western blotting and immunocytochemical staining. Results Compared with negative control group,after treated with 20 μg?L-1 PPD for 48 h,the apoptotic rate of Siha cells was increased (P<0.05),the transcription and protein expressions of caspase-9 and caspase-3 were up-regulated(P<0.01),and the content of cleaved caspase-3 was increased(P<0.01). Cell immunochemistry detection showed the  brown particles in the experimental  cells in 20  μg?L-1 PPD group. Conclusion PPD may induce the apoptosis of Siha cells,and the mechanism may be related to the up-regulation and activition of caspase family.

Abstract:Objective
To investigate the combination effects of rapamycin(RAPA) and adriamycin  (ADM) on the proliferation and migration of hepatoma cell BEL-7402，and furthermore  analyze the possible mechanisms. Methods The 　hepatoma cell BEL-7402 in logarithmic growth phase were  selected as the research object，and  were randomly divided into five groups：blank control group，vehicle group，RAPA group ，ADM group and combination group.The cell proliferation in 6 d in  five groups was detected by MTT，the cyclin D1 mRNA and MMP-2 mRNA expressions in various groups were detected by RT-PCR，and the cyclin D1 protein expression was detected by Western blotting. Results From the 3rd day to the 6th day of the cell culture，the cell proliferation rate in combination group was lower than those in control，vehicle，RAPA and ADM groups（P<0.01）.The expression of cyclin D1 mRNA in combination group（0.63）was lower than those  in control(1.02),RAPA（0.77）and ADM（0.76） groups.The expression of MMP-2 mRNA in combination group（0.88）was lower than those in control(1.98),RAPA（1.40） and ADM （1.28） groups.The expression of cyclin D1 protein in combination group(0.33) was lower than those in control(0.92),RAPA（0.75）and ADM （0.74） groups. Conclusion The combination treatment of RAPA and ADM can improve the sensitivity of hepatoma cell BEL-7402 to ADM,the mechanism perhaps lies in the combination of the two drugs can inhibit the expressions of cyclin D1 mRNA,MMP-2 mRNA and cyclin D1 protein more significantly.

Abstract:Objective To observe the intestine absorption kinetics of berberine (Ber) in rats and the effects of sodium caprate(SC) on Ber in intestinal absorption. Methods Male Wistar rats were used for the experiment. The in situ circular perfusion model was used to investigate the effect of SC on the intestinal absorption of Ber. The concentration of Ber was detected with  HPLC.The  activity of LDH was measured to evaluate the toxic effect of SC on mucous membrane cells. Results The absorption of Ber in the small intestine was poor. The uptake of Ber presented dose-dependent manner. There was no significant difference on Ka among these groups (P>0.05). SC could significantly enhance the absorption amount of Ber in the small intestine (P<0.05). SC showed no significant effect on Ka of Ber(P>0.05). The LDH activities in circulating fluid  in Ber and SC+Ber groups  had no significant difference compared with control group (P>0.05). Conclusion The absorption of Ber in the small intestine is poor and complied with the passive diffusion mechanism and first order kinetics. SC can significantly enhance the absorption of Ber in the small intestine and is a safe,effective absorption accelerator.

Abstract:Objective
To explore the effects of angiotensin Ⅱ(AngⅡ)on the synthesis and release of plasminogen activator inhibitor 1(PAI-1) and tissue plasminogen activator(tPA) through modulating extracellular signal regulated kinase(ERK) activated with AngⅡtype 1 receptor(ATlR) in rats with acute myocardial infarction(AMI). Methods 24 Sprague-Dawley rats were randomly divided into 3 groups:sham operation group(n=8),AMI group(n=8),and PD98059 group(n=8). The rat model of AMI was established by ligating left anterior descend coronary artery. On the 14th day after AMI,the tissue slices of myocardium were prepared and the histopathological changes of myocardium were observed with microscope;the AngⅡ levels in plasma and aortic tissue were measured by radioimmunoassay;the PAI-1 and tPA activities were measured by spectrophotometric assay;the expressions of p-ERK1/2 and ATlR proteins in the homogenized aortic tissue were examined by Western blotting in rats in various groups.Results ①The HE-dyed tissue slices of myocardium showed that the myocardium was normal(cardiac muscle fibers to line up in order and nucleus being rule) in sham operation group but abnormal(the disruption,cavity and dissolution of cardiac muscle fibers,the lysis of nucleus,the proliferation of fibrous connective tissue and the infiltration of inflammatory cells) in AMI group,and  the damaged myocardium after AMI was remarkably improved in PD98059 group. ② Compared with sham operation group，the AngⅡlevels and PAI-1 activities in plasma and incubation solution for aorta were significantly increased(all P<0.05)，but the activity of tPA and the ratio of tPA to PAI-1 in plasma and incubation solution for aorta were decreased remarkably in AMI group(all P<0.05). ③Compared with sham operation group，the expressions of p-ERK1/2 and ATlR proteins in homogenized aortic tissue were significantly increased(all P<0.05). ④The plasma AngⅡ level and PAI-1 activity，aortic AngⅡ level and  PAI-1 activity were all closely related to the expressions of p-ERK1/2 and ATlR proteins in homogenized aortic tissue in various groups(r=0.85－0.94,all P<0.01).⑤Compared with AMI group,the AngⅡ levels in plasma and homogenized aortic tissue,and the activity of PAI-1 in plasma and incubation solution for aorta,as well as the expression of p-ERK1/2 and ATlR proteins in the homogenized aortic tissue were markedly reduced(all P<0.05),but the activity of tPA and the ratio of tPA to PAI-1 were enhanced notably in plasma and incubation solution for aorta in PD98059 group(all P<0.05). Conclusion In the condition of AMI,Ang Ⅱcould enhance the synthesis and release of PAI-1 but inhibit the synthesis and release of tPA through activating ATlR to trigger activation of ERK.

Abstract:Objective
To investigate the activation of p38MAPK protein in cultured cortical neurons after NMDA injury and intervention effect of W-7. Methods The neurons were identified by polyclonal antibody against neuron specific enolase (NSE).The primary cortical neurons cultured for 7 d were randomly divided into 5 groups:control group;NMDA-injured group;three-doses of W-7 pretreatment groups. The cortical neurons were pre-cultured with regular media including different doses of W-7 respectively as 25,50 and 100 μmol.L^-1 for 24 h before exposed to NMDA (50 μmol.L^-1). The p38MAPK protein expression in cultured cortical neurons was detected by  immunocytochemiscal staining and Western blotting. Results  A large number of hippocampal neurons began to adhere to cover the glasses 6-12 h after culture. They showed different shapes after clinging to the plate. Their processes connected into nets and they were different in length and thickness. The proportion of positive neurons was 90.86%.Compared with control group,the expression of p38MAPK in cultured neuron was remarkably up-regulated in NMDA injured group by immunocytochemistrical staining  and up-regulated in NMDA injured group by Western blotting (P<0.05),it was significantly down-regulated in W-7 pretreatment groups compared with NMDA group (P<0.05 or P<0.01). Conclusion NMDA could up-regulate the expressin of p38MAPK in cultured cotical neurons,and W-7 could down-regulated the expression of p38MAPK.

bstract:Objective
To obain the C terminal gene encoding human mannose-binding lectin associated serine protein kinase-2(MASP-2),and express human MASP-2 C-terminal fragment and clinical application in E.coli and lay foundation for preparation of monoclonal antibodies and clinical application in relevant diseases. Methods The cDNA of human MASP-2 C-terminal was amplified from total RNA extracted from human fetal liver tissue with RT-PCR,and was cloned into pGEX-6p-2 vector and identified by restrictive digestion analysis and sequencing. Results The cDNA encoding human MASP-2 C-terminal was isolated,linked with pGEX-6p-2 vector and transformed into E.coli XL1-blue. The restriction map analysis and sequencing results were consistent with the sequence of MASP-2 C-terminal cDNA that  reported in GenBank. Conclusion The human MASP-2 C-terminal cDNA is successfully cloned and expresses in E.coli.

Abstract:Objective
To study the inhibitory effects of cadmium chloride (CdCl2) on SMMC-7721 cells in hepatocarcinoma    transplanted nude mice and influence in mitochondrial enzymes and clarify their mechanisms. Methods The hepatocarcinoma transplanted nude mouse model were established in vivo. The mice in negative control group were administered with sodium chloride,the mice in positive control group were administered with 25 mg.kg^-1 　5-Fu，and the mice in Cdcl2 groups were administered with   0.5,1.0,2.0 mg.kg^-1 1 CdCl2,respectively. The anti-tumor effects of CdCl2,the changes of serum ALT and AST levels, the activities of LDH and ATPase in mitochondria were detected 10 d after administration. Results Compared with positive control group,the inhibitory rate of tumor tended to increase along with the elevating of CdCl2 dose,but there were no statistical significances (P>0.05). The organ indexes in all CdCl2 groups didn’t change greatly,but the kidney organ indexes  in CdCl2 groups were higher than that in positive control group (P<0.05). Compared with  negative control group,the level of ALT and  the activity of LDH in CdCl2 groups were significantly decreased(P<0.05),and the level of AST in 0.5 and 1.0 mg.kg^-1 CdCl2 groups were also  significantly decreased(P<0.05).The activities of Ca ²⁺ -Mg ²⁺ -ATPase in 1.0 and 2.0 mg.kg^-1  CdCl2 groups were significantly increased compared with  negative control group(P<0.05),the activities of Na ⁺ -K⁺-ATPase in 0.5 and 2.0 mg?kg-1 CdCl2 groups were  significantly increased  compared with  positive control group(P<0.05). Conclusion CdCl2  may interfere the energy metabolism of mitochondria and  inhibit the growth of  the transplanted hepatocarcinoma through mitochondria way.

Abstract:Objective
To explore the effects of high dose vitamin E(VE) on lipid metabolism in rats and provide experimental basis for intaking amount of VE. Methods 28 adult female Wistar rats were randomly divided into four groups including control,low dose VE (400 mg.kg^-1),medium dose VE(800 mg.kg^-1),and high dose VE (1 600 mg.kg^-1)  groups. Administered by intragastrical route for 16 d,the blood samples were collected and the contents of TC,TG and HDL-C were measured,and the ratios of organ over body weight in liver,kidney and spleen were calculated. Results  Compared with control  group,the water volume and food consumed by rats in  VE groups were less;the rats were less active.Compared with control and low dose VE groups,the body weights of rats in medium and high dose VE groups were decreased(P<0.05). There were no significant differences in the ratios of organ over body weight in liver,kidney and spleen between control  and VE groups（P>0.05）. Compared with control group,the serum contents of TC,TG,and HDL-C in medium and high dose VE groups  were significantly decreased(P<0.05). Compared with low dose VE group,the contents of serum TC,TG,and HDL-C 　in medium and high dose VE groups  were significantly decreased (P<0.05). There were no significant differences in serum contents of TC,TG,and HDL-C between medium and high dose VE groups（P>0.05）. Conclusion High dose VE intake can produce harmful effects on rats. Overdose of VE intake through oral administration will decrease the content of HDL-C and has adverse effects on lipid metabolism.

Abstract:Objective To investigate the effects of E6-AP on cell proliferation and cell cycle of human prostate cancer cells and elucidate the role of E6-AP participating in androgen receptor signal pathway and in the progress of prostate cancer.  Methods tTA and E6-AP overexpression LNCaP cell line was established by electroporation transfection. The cell viability was analyzed by MTT assay and cell cycle distribution was detected by flow cytometry after propidium iodide staining using the stable cell line under the regulation of Dox. Results LNCaP cell line overexpressing E6-AP was established successfully. Compared with untreated LNCaP cell and Dox treated E6-AP stable cell line,the total number and the proportion in S phase of Dox treated E6-AP stable cells were increased significantly（P<0.01）. Conclusion Overexpression of E6-AP induces proliferation of LNCaP cells and increase the number of cells in S phase,indicating that E6-AP is involved in androgen receptor signal pathway.

Abstract:Objective
To observe the effect of BCG-derived HSP70（BCGHSP70） on the immunogenicity of tumor cell lysate and  provide basis for the preparation of TCL-based tumor vaccines. Methods  B16TCL was prepared from B16 melanoma cells by freeze-thaw,then directly mixed with the recombinant BCGHSP70 in vitro to generate BCGHSP70-B16TCL. C57BL/6 mice were randomly divided into 4 groups:BCGHSP70-B16TCL group,PBS group,BCGHSP70 group and B16TCL group. On days 1 and 14,the mice were injected subcutaneously with BCGHSP70-B16TCL,PBS,BCGHSP70 or B16TCL, respectively. On day 21,the mice were scarified and the splenocytes from each group were tested for their proliferative response to the stimulation of B16TCL in vitro by MTT assay,and the stimulation index (SI) of splenocytes was calculated. Results B16 melanoma cells were lysed completely by freeze-thaw,and abundant proteins were observed in the B16TCL. BCGHSP70-B16TCL immunization resulted in a significant increase in the size of spleens. The SI of splenocytes was higher in BCGHSP70-B16TCL group than those in PBS group (P=0.00),B16TCL group (P=0.01) and BCGHSP70 group (P=0.00). Conclusion BCGHSP70 could enhance the immunogenicity of B16TCL,indicating that BCGHSP70 might be used as an effective adjuvant for the TCL-based tumor vaccine.

Abstract:Objective
To investigate the expression level of STAT3 protein in human breast cancer tissues and corresponding adjacent normal mammary tissues,and clarify the relationship between STAT3 expression and clinical characters  in patients with breast cancer. Methods The expressions of STAT3 protein in 67 human breast cancer tissues  and the corresponding adjacent normal mammary tissues were detected by immunohistochemical technique.The expressions of STAT3 protein in patients with different clinicopathological parameters were analyzed. Results The expression rate of STAT3 protein in 67 human breast cancer tissues was 62.69% (42/67) which was significantly higher than that in normal  mammary tissues (25.37%,P<0.001). The expression level of STAT3  had no correlation with the age,tumor size and histological type in patients with breast cancer(P>0.05),but the expression of STAT3 protein was positively correlated with clinical stage,lymph node metastasis and survival time in  patients with breast cancer. The mean survival time of the patients with high expression of STAT3 protein was shorter than that of the patients with low expression of STAT3 protein(P<0.05). Conclusion STAT3 protein over-expression may play a promotion  role in the tumorigenesis and development of breast cancer,and may be an important prognostic factor in breast cancer.

Objective To detect the expressions of  focal adhesion kinase (FAK) and extracellular signal-regulated protein kinase（ERK）in  salivary adenoid cystic carcinoma (SACC) cells SACC-LM and SACC-83 and explore their relationship with occurrence of lung metastasis of SACC. Methods Western blotting was applied to detect the  protein expressions of  FAK and ERK  in SACC-LM and SACC-83 in G1/S phase and the enzyme activities of FAK and ERK,the results were quantified. Results The activities of FAK and ERK in SACC-LM cells were higher than those in SACC-83 cells（P<0.01）,the activity of FAK in SACC-LM cells had positive correlation with the activity of ERK(r=0.62，P<0.05).The Western blotting results showed  that the  protein expression levels of FAK and ERK in SACC-LM cells were higher than those in  SACC-83 cells（P<0.05）. Conclusion FAK and ERK may be involved in the occurrence and development process and metastasis signal conduction passage of SACC,the protein expressions of FAK and ERK concerns with the variation behavior of SACC.

Abstract:Objective To detect the mutation of  acid-resistant gene gluA in the fluoride-resistant strain of streptococcus mutans and clarify the  acid-resistant change of fluoride-resistant strain.  Methods Streptococcus mutans were artificially induced  into fluoride-resistant strain of streptococcus mutans in vitro.The genome of fluoride- resistant strain of streptococcus mutans was obtained by  alkaline method;in accordance with  primer design principles,according to the published GenBank sequence of streptococcus mutans gluA, a pair of  primers were designed  for PCR.The PCR products were cloned into pMD18-T vector and the recombinant plasmid was constructed.The insertion fragment was identified with restrictive enzyme and sequenced. Results By BLASTN comparison,the sequence of  acid-risistant gene gluA of the fluoride-resistant strain of streptococcus mutans was the same with that reported in GenBank. Conclusion The acid-resistant gene gluA of the fluoride-resistant strain of streptococcus mutans has not been any mutation which has no impact on the acid-resistant character of fluoride-resistant strain of streptococcus mutans.

Objective To evaluate the effects of platelet-rich plasma (PRP) on proliferation of human dermal fibroblasts (hDFbs) in vitro and investigate the mechanism of PRP promoting wound healing. Methods hDFbs and PRP  were separated from a healthy adult donor. PRP was prepared by a double centrifugation technique. In the proliferation experiment ainverted phase contrast microscope was used to observe the growth of cells after treated with different concentrations PRP,such as 0,12.5%,25.0%,50.0% and 100.0%. Immunocytochemistry was used to evaluate the expressions of platelet-derived growth factor (PDGF) treated with 50.0% PRP in different volumes.Fluorescent coloration was used to observe the  proliferation of the cells which adhered to the titanium material. Cell cycle of hDFbs was detected by flow cytometry. The cell proliferation was also evaluated by CCK-8 assay. Results The microscope investigation showed that the number of cells in 100%PRP group was higher than those in other groups. The significant increase in PDGF expression was observed when treated with  30 μL PRP，but the IOD in 10 μL PRP group was higher than that in 20 μL PRP group(836.27±21.15  vs  794.35±30.26，P<0.05). The fluorescent coloration showed that PRP  improved the cell quantity on the material compared with  control group. The flow cytometry  showed that PRP  stimulated the cells to enter the S phage to copy DNA. In the 2nd day after treated with PRP the percent of the cells  in S phage in  PRP group was  higher than that in control group (34.41%  vs  22.00%,P<0.05);In the 8th day the  percent of  the cells  in  G0G1 phage  was  higher than that in control group (95.07%  vs  89.70%,P<0.05 ).The result of  CCK-8 showed  that the  absorbency number in 100.0% PRP group was significantly higher than that in 12.5%group (1.13±0.05  vs  0.75±0.04,P<0.05) in the 4th day. Conclusion High concentration PRP has a positive influence on hDFbs proliferation, but it isn’t dose-dependent. An appropriate concentration PRP can promote the proliferation of hDFbs.

Objective
To compare the flexural strength of the four kinds of room curing denture materials of polymethyl methacrylate(PMMA),and  observe the physical conformation section through confocal laser scanning microscope (CLSM) in order to find the relationship between the macro-bending force and the microstructure. Methods Four room curing PMMA materials  were produced by  Tokuyama(hard-liner type),Nisshin(fiber type),Heraeus(fiber type), and Shanghai dental (simple type). Each of the room curing PMMA was prepared by a group of 10 specimens. According to ISO1567,room temperature 20℃ and vendors powder liquid ratio with the stainless steel molds were performed to prepare samples,flexural strength was measured on the test machine. The cross-section was analyzed with CLSM. Results The  flexural strengths of four kinds of room curing PMMA  were (41.00±12.22),(87.80±14.82),(76.30±7.51), and (81.20±8.93) MPa,respectively;there wre significant differences in the flexural strengths between  Tokuyama and Nisshin,Heraeus(P<0.05), there was significant difference between Shanghai dental and Tokuyama  (P<0.05),but there was no significant difference  between Shanghai dental and Nisshin,Heraeus (P>0.05).In the cross-section image of Nisshin group the complete degree  and the number of micro-cracks  were significantly higher than others,there was no significant difference between Heraeus and Shanghai dental,Tokuyama was the lowest. Conclusion Tokuyama has  the lowest flexural strength,Nisshin maximum,Heraeus similarity with Shanghai dental. When the flexural  strength of the material is stronger,there are more micro-cracks  from the cross-section.

Abstract:Objective
To detect the Notch signaling pathway-related protein expression levels in human renal cell carcinoma (RCC)   tissues  and explore the  relationship between Notch signaling pathway molecule and occurrence and  development of RCC.Methods 6 cases of RCC diagnosed by pathology detection were obtained by surgical methods.The Notch signaling pathway receptor and ligand protein levels in RCC tissues and adjacent tissues were detected with Western blotting. Results Compared with adjacent tissues, the expression levels of the receptor of Notch signaling Notch-1 and the ligand Jagged-1 in RCC tissues were  significantly reduced（P<0.05）,the expression levels of the  receptor Notch-2 of the  Notch signaling pathway and the ligand Jagged-2  in RCC tissues were significantly  increased（P<0.05）.Conclusion The expression of N
otch signaling pathway protein in RCC has apparente change,Notch signaling pathway may be involved in the occurrence and development of RCC.

Objective
To investigate the expressions  of platelet-derived growth factors(PDGFs) in primary adenocarcinoma of stomach tissues,and clarify their relationships with clinicolpathological features of primary adenocarcinoma of stomach. Methods The mRNA expressions  of PDGF-A，B，C，D were assessed by reverse transcription-polymerase chain reaction（RT-PCR）in 58 cases of gastric cancer and adjacent carcinoma tissues. Immunohistochemical staining was used to examine the expression of PDGF-D protein in gastric cancer and adjacent normal tissues. Results The positive expression rates of PDGF-A,B,C,D mRNA were 39.66%,22.41％,31.03%, and 98.28％ in gastric cancer tissues and 12.07％,10.34%, 17.24%, and 72.41% in adjacent normal tissues,respectively.The positive  expression rate of PDGF-D mRNA  in gastric cancer tissues was significantly higher than that in adjacent normal tissues（P<0.05）.The expression of PDGF-D mRNA was significantly higher than other PDGF mumbers in gastric cancer tissues (P<0.0１).The PDGF-D protein was present in membrane/ cytoplasm and the difference between gastric cancer and adjacent normal tissue was statistically significant (P<0.05). The PDGF-D mRNA expressions in groups with lymph-node metastasis,distant metastasis and relapse were higher than those in the groups without metastasis and relapse (P<0.05）. Conclusion PDGF-D is the most important mumber of PDGFs and has a close relationship with human gastric cancer. It can be used as a prognostic marker of gastric cancer in clinical practice.

Objective
 To compare the difference between the measurement results of  the  cervical  pedicle specimens  and CT image，and provide the basis for clinical cervical screw internal fixation operation. Methods Twenty-seven Chinese adult cadaver cervical specimens including C3 to C7 vertebrae were measured by a digital calipers and  CT image，containing pedicle height（PH,PH′）,pedicle width(PW,PW′),total pedicle length(TL,TL′)and two pedicle lengths（PL1,PL2;PL1′，PL2′）.The results of specimens and  CT images were compared. Results Different cervical vertebra in the same side of specimens or CT images,PW(PW′):C3,C4＜C5，C6（P<0.05)，C5，C6＜C7（P<0.01) ； PH(PH′):there were no significant differences；TL,PL1，PL2 (TL′，PL1′,PL2′):there were no marked differences.In the same cervical vertebra of the specimens or CT images,PW(PW′)＜PH(PH′) (P<0.01﹚，PL1(PL1′)＜PL2(PL2′) (P<0.01).Conclusion The results of measurement by CT images are not markedly different from that of specimens. CT image measurement is available before cervical screw internal fixation operation.

Objective
To determine the serum levels of a proliferation-inducing ligand(APRIL) in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis(RA) and analyze its correlations with immunological parameters and its roles in the development of SLE.Methods The levels of  APRIL  in 48 patients with SLE,16 patients with RA and 16 healthy controls were determined by enzyme linked immunosorbent assay (ELISA),its correlations with SLEDAI and immunological parameters were analyzed.Results The serum levels of APRIL in SLE and RA patients were significantly higher than  that in healthy controls （P<0.01）,and the serum level of APRIL in SLE  patients were significantly higher than that in RA patients（P<0.01）. The serum level of  APRIL in SLE patients with positive anti-Sm antibody was significantly higher than that in the negative ones（P<0.05）.The serum level of APRIL in SLE patients with positive anti-U1RNP antibody was significantly higher than that in the negative ones（P<0.05）.While the serum level of APRIL in SLE patients  didn’t have significant differences between the patients with positive anti-SSA /SSB antibody,anti-ACL antibody,anti-dsDNA antibody and the negative ones.The serum level of APRIL in SLE patients was  negatively correlated with the complements C3 and C4 （r1=－0.819，P1＜0.01；r2=－0.549，P2＜0.01）,but not significantly correlated with SLEDAI and other immunological parameters.Conclusion The serum level of APRIL in SLE patients is specially increased,it may have an important effect on the development of SLE and may be correlated with the activity of SLE .

To study the clinical phenotypes and the   molecular biological characteristics  in a family with nonmuscle myosin heavy chain 9 related disease（MYH9-RD）and reveal the molecular pathogenesis mechanism of MYH9-RD .Methods A screen inclinding clinical and laboratory features was made for this family,the platelet count and peripheral blood morphology were observed  by automatic blood cell counter and light microscope,respectively. Polymerase chain reaction and direct sequcing method were used to analyze the mutation of MYH9 gene. Results There were 15 patients with MYH9-RD in this family,all the patients not only had thrombocytopenia,giant platelets and inclusion bodies in granulocytes,but also had easy bruising and mild to moderate bleeding tendency;Moreover,some even suffered from serious phenotypes,such as leucocythemia,glaucoma,cataract,proteinuria,abnormal hepatic function,hyperlipemia and disordered action of heart etc. No pathgenic mutation was detected in the exon or franking regions of MYH9 gene of patients.Conclusion The family patients are diagnosed as MYH9-RD by clinical and laboratory features,and the complicated phenotypes of the patients are may be related to the no pathgenic mutation in the 40 exon of MYH9 gene of patients.

Abstract:Objective
To explore the correlation between the E211 G>A androgen receptor(AR) gene polymorphism and female postadolescent acne of Han Chinese,and lay the foundation for genetic studies of female postadolescent acne of Han Chinese.  Methods The female postadolescent acne patients(n=79) and the healthy people(n=80) were individed into female postadolescent acne group and control group. The polymorphism was investigated by PCR using DNA from peripheral blood lymphocytes. The transition(G>A) in the risk allele(A) produced a new recognition site for the estriction endonuclease StuⅠ. Three genotypes of AR-E211 G>A gene (GG,GA,AA) were determined,and   the differences in the frequencies of the genotype and the allele between female postadolescent acne group and control group were observed. Results There was a significant difference in the frequency of the genotype GA+AA in the AR-E211 genetic polymorphisms between female postadolescent acne patients and healthy control (P=0.028,OR=0.937,OR 95% CI=0.885－0.992);and there was also a significant difference in the allele (A) frequency between female postadolescent acne patients and healthy control (P=－0.029,OR=0.968,OR 95% CI=0.941-0.996).Conclusion The high frequency of A allele in the functional G1733A polymorphism of AR-E211 is a decreased risk factor for female postadolescent acnein Han Chinese.

Objective
To investigate the correlation between the polymorphisms of 4 SNPs of Tim-3 gene and susceptibility of rheumatoid arthritis (RA) in Ningxia Hui population and provide theory basis for taking precautions against RA. Methods Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP),specific sequence primer-polymerase chain reaction (SSP-PCR) genotyping method were carried out to examine the SNPs at -574, -882,-1541 and 4259 sites of Tim-3 gene,including 111 healthy individuals and 113 RA patients in Ningxia Hui population. Results Both genotypic frequency and allelic frequency at -574,-882,-1541 sites of Tim-3 gene had no significant difference（P>0.05）,while both genotypic frequency and allelic frequency at 4259 site had significant difference between patient group and normal group（P<0.05）. The frequency of allele G in patient group was significantly higher  than that in normal group（P<0.05).Conclusion -574,-882,-1541 and 4259 sites of Tim-3 gene have SNP variations,and 4259 T>G mutation gene is related to the pathogenesis of RA in Ningxia Hui population.

Objective
To explore the efficacy and influencing factors of interventional therapy for chronic atherosclerotic occlusive disease of the lower extremity. Methods 56 cases of simple atherosclerotic occlusive disease of lower extremity were divided into 3 types according to the location of occlusion,including 17 cases of abdominal aorta-iliac artery,18 cases of iliac-femoral artery,and 21 cases of femoropopliteal inferior genicular artery. Catheter and guide were combined with blunt dissection and subtle banding were used to dredge the occluded vessels,and the stents were implanted when interlayer appeared. Results  After the treatment,46 cases of occlusion were dredged (82.1%),6 cases remained occluded with the same symptoms (10.7%),and 4 cases had severer symptoms (7.1%). Among the 46 successful cases,there were 16 cases of abdominal aorta-iliac artery (94.1%),16 cases of iliac-femoral artery (88.9%) and 14 cases of femoropopliteal inferior genicular artery(66.6%). There was no statistical difference between the abdominal aorta-iliac artery cases and iliac-femoral artery cases (P>0.05),but the rate of abdominal aorta-iliac artery was higher than that of femoropopliteal inferior aorta-iliac artery (P<0.05).In the successful cases,9 cases of abdominal aorta-iliac artery (52.9%),10 cases of iliac-femoral artery (55.5%) and 7 cases of femoropopliteal inferior genicular artery (33.3%) had 5-10 cm of occlusion,there was no significant difference between the first two types(P>0.05),but there were significant differences between the first two types and the third type (P<0.05).Compared with the 14 dredged cases (71.4%),7 cases (33.3%) of undredged femoropopliteal inferior genicular artery cases had richer compensatory circulation (P<0.05). Conclusion It is safe and effective to treat simple atherosclerotic occlusive disease of the lower extremity with interventional therapy. The location and length of occlusion and the abundance of compensatory circulation have effects on the therapy.

Objective
To study the changes of the expressions of VEGF,bFGF and CD34 in vulvar white lesion tissues  before and after focusing ultrasound therapy and clarify the mechanism of focusing ultrasound in treatment of vulvar white lesion. Methods The expressions of  VEGF,bFGF and CD34 in 30 cases of vulvar white lesion tissues,15 cases one month after  treatment and 8 cases three months after  treatment were detected by  SP immunohistochemistry method. The positive expressions of VEGF,bFGF and CD34 in vulvar white lesion tissues in three groups before and after  treatment were compared.
Results The expressions of VEGF and bFGF in vulvar white lesion tissues after treatment were higher than before treatment(P<0.05).The expression of CD34 in vulvar white lesion tissues had no significant difference between before and after treatment (P>0.05). Conclusion Focusing ultrasound therapy could increase the expressions  of VEGF and bFGF in vulvar white lesion tissues and improve the structure and function of nerve and capillary.

Abstract:Objective To evaluate the potential sparing effects of dexmedetomidine(Dex) on propofol and fentanyl and suppression of sympathoadrenal responses to tracheal intubation and extubation and the influence in recovery from anesthesia.  Methods  A total of 24 patients (ASA Ⅰ-Ⅱ) undergoing selective operation practicing general anesthesia were randomly divided into 2 groups:Dex group (n=12) and control group (n=12).30 min before induction the patients in Dex group received 1 μg/kg Dex diluted to 10 mL over 10 min by pumped infusion and the patients in  control group simply recieved normal saline at the same way.The patients in both groups were pumped 0.4 mg?kg-1?min-1 propofol at the beginning of induction.When holding up jaw without bodymove,the patients received 1 μg/kg fentanyl and 0.6 mg/kg rocuronium,and were endotracheally intubated 1.5 min later.After being intubated successfully they received sevoflurane by inhalation instead of propofol. At the same time the patients in Dex group received 0.3 μg/kg/h Dex till the operation finished and the patients in control group received normal saline at the same way. Sevoflurane ceased at the end of surgery.The dose of propofol duing induction and fentanyl during the entire operation were recorded. The HR and MAP of the patients were recorded when they entering the operation room(T0),being inserted laryngoscopy(T1),being endotracheally intubated(T2),opening their eyes after operation(T3),being extubated(T4) and 10 min after being extubated(T5).The postoperative respiration recovery time,time to open eyes and extubation time after operation were recorded.  Results Compared with control group,the dose of propofol( mg/kg) during inducion was decreased by 21%（P<0.01）and the dose of fentanyl during the entire operation was decreased by 38% in Dex group (P<0.01).The MAP and HR in control group were rised significantly at T1,T2 and T4 compared with T0(P<0.01).The MAP and HR at T1-T6 in Dex group had no significant change compared with T0.The MAP and HR in Dex group were decreased obviously at T1,T2 and T4 compared with control group(P<0.01).The postoperative respiration recovery time,time to open eyes and extubation time had no  significantly difference between two groups（P>0.05）.  Conclusion Dex may reduce the dose of propofol duing induction and fentanyl during the entire operation and have hemodynamic-stabilizing effects during tracheal intubation and extubation while not prolonging the recovery time from anesthesia .

Abstract:Objective To evaluate the microleakage degree of  complete cast-metal crowns cemented onto teeth that were rebuilt with different core materials and   provide some evidences for clinical application. Methods Ninety extracted molar teeth were randomly divided into nine groups as follows: amalgam / super bond C&B,amalgam/ zinc polycarboxylate,amalgam/ glass-ionomer CX,gold/ super bond C&B,gold/ zinc polycarboxylate,gold/ glass-ionomer CX,composite/ super bond C&B, composite/ zinc polycarboxylate,composite/ glass-ionomer CX. Tooth preparations for complete veneer cast crowns were completed. A classⅡcavity preparation with fixed dimension was made in each tooth. Three different core materials(composite resin,amalgam,cast-gold) were filled in them. The artificial crowns were cast and cemented onto teeth by using three different cements(composite resin super bond C&B,glass ionomer CX,zinc polycarboxylate).The specimens were thermocycled and dyed. They were then embedded in epoxy resin and sectioned. The degree of microleakage was evaluated and scored.  Results In super bond C&B group,the three different cores showed no significant difference in core base microleakage. In zinc polycarboxylate group and glass-ionomer CX group,the core base microleakage in amalgam and composite were significantly different from those in gold(P<0.05). The crown microleakage degree of composite resin was lower than those of glass ionomer and zinc polycarboxylate (P<0.05).Conclusion The  microleakage degree of composite resin is less than those of glass ionomer and zinc polycarboxylate,and the microleakage of amalgam and composite resin is also lower than that of cast-gold core materials.

Abstract:Objective To evaluate the regional left ventricular systolic function of patients with late-stage liver cirrhosis by application of vector myocardial strain and strain rate imaging (VSI),and discuss the changes of the regional left ventricular systolic function of patients with late-stage liver cirrhosis. Methods The longitudinal peak systolic strain rates(L-SRs),radial peak systolic strain rates(R-SRs) and circumferential peak systolic velocity(C-Vs) in 30  patients with late-stage liver cirrhosis (patient group) and 30 cases of age-matched normal persons (control group) were detected by VSI technology. Results The  L-SRs of mid-anteroseptal,apical-anterior and apical-septal in patient group were reduced compared with control group (P<0.05);the R-SRs of mid-anteroseptal,apical-anterior and apical-septal in patient group were reduced compared with control group (P<0.05);the C-Vs of mid-anterior and mid-anteroseptal in the cirrhosis group was reduced compared with control group (P<0.05). Conclusion The regional left ventricular systolic function of patients with late-stage liver cirrhosis has already reduced before the global left ventricular systolic function has not significantly affected.

Abstract:Objective To study the value of apparent diffusion coefffcient (ADC) value in diagnosis of glioma before operation by analyzing the differences of ADC value between low grade glioma and high grade glioma. Methods The imagines of 14 cases of low grade gliomas (Ⅰ,Ⅱ grade) and 20 cases of high grade gliomas (Ⅲ,Ⅳ grade) with glioma histologically proved including conventional MRI,enhanced scanning and DWI were retrospectively analyzed. The ADC map in the station building was constructed and the ADC values of tumor substantial part,peritumoral edema,cystic necrotic area and the corresponding contralateral normal brain were measured. The relationship between the ADC value about the measurement area and the grade of glioma was analyzed.
Results The ADC value of tumor substantial part of low grade glioma ［(1.47±0.18)×10^-3 mm²/s］ was significantly higher than that of high grade glioma ［(0.97±0.18)×10^-3 mm²/s］ (P<0.01)，the ADC values of the corresponding contralateral brain white matter were (0.76±0.06)×10^-3 mm²/sand (0.75±0.06)×10^-3 mm²/s;and the ADC value of tumor peritumoral edema of low grade glioma ［(1.61±0.19)×10^-3 mm²/s］ was significantly higher than that of high grade glioma ［(1.26±0.14)×10^-3 mm²/s］ (P<0.01),the ADC values of the corresponding contralateral brain white matter were (0.74±0.04)×10^-3 mm²/s and (0.77±0.06)×10^-3 mm²/s. The ADC value of cystic necrotic area of low grade glioma ［(1.88±0.11)×10^-3 mm²/s］ was higher than that of high grade glioma ［(1.34±0.14)×10^-3 mm²/s］ (P<0.01),the ADC values of the corresponding contralateral brain white matter were[JP3](0.75±0.08)×10^-3 mm²/s and (0.73±0.06)×10^-3 mm²/s. The ADC values of tumor substantial part,peritumoral edema,cystic necrotic area were inversely associeated with the degree of malignancy of the gliomas(the r values were －0.821,－0.726 and －0.919,respectively,P<0.01). Conclusion The ADC values of tumor substantial part,peritumoral edema,cystic necrotic area between low grade glioma and high grade glioma have significantly differences,the measurement of ADC value play an important role in diagnosis of glioma grading before operation.

Abstract:Objective To evaluate the clinical value of speckle tracking imaging（STI）for the measurement of left ventricular short-axis views regional rotation in patients with heart failure （HF）. Methods High frame rate two-dimensional images from the left ventricular short-axis views in 31 patients with CHF(CHF group) and 32 healthy　controls(control group)  were recorded. The regional rotation（Rot）was measured in the left ventricular short-axis views;the peak endocardium  rotation (endo-rot),peak epicardium rotation (epi-rot),peak bulk rotation(bulk-rot),and peak mural torsion(mural-tor) were measured separately. Results Compared with control group,the regional Rot of each segment in patients with CHF was singnificantly decreased(all P<0.01),the peak endo-rot,peak epi-rot,peak bulk-rot,and peak mural-tor were aslo significantly decreased（all P<0.05） in patients with CHF. The left ventricular torsion of patients with CHF was significantly lower than that in control group（P<0.01）. Conclusion  STI can exactly evaluate the characteristics of left ventricular rotation and the heart function in patients with CHF.

Abstract:Objective To culture and identify chemotherapy microspheres (CMSs) isolated from tumor tissues of breast cancer patients after receiving neoadjuvant chemotherapy and provide the theoretical basis for a new method to obtain breast cancer stem cells. Methods After CMSs were isolated from   the breast cancer tissues,the experiments were conducted as follows:① CMSs were plated in DMEM/F12 media containing growth factors and the growth process of CMSs was observed;② Sphere formation assay was performed to determine the self-renewal ability of chemotherapy microspheres-derived cells (CMSDCs);③ The differentiation ability of CMSDCs was observed by culturing them in DMEM/F12 containing 5% fetal bovine serum media;④ The expressions of CK14,CK18 and CK19 of CMSDCs and their differentiated cells were detected by immunocytochemistry;⑤ The ratios of ALDH1+ cells of CMSDCs and their differentiated cells were evaluated by flow cytometry (FCM);⑥ The CMSDCs were implanted into NOD/SCID mice to assess their tumorigenic potential. Results The CMSs could continue to grow in serum-free media and the necrotic cells were distributed over the surface layer of the CMSs after 3 d;The CMSDCs could form new CMSs with strong refraction by serial sphere formation assay;The CMSDCs could differentiate to monolayer of spindle-shaped cells in the media  supplemented with serum;The CMSDCs expressed the breast stem cell marker  CK19,but not CK14 and 18,which marked the myoepithelial and luminal cells,respectively;The ratio of ALDH1+ cells of the CMSDCs accounted for 17.41%-23.57% of all the cells，but which of their differented cells accounted for only 0.50%-1.28% of the total cells;New tumor identical with the primary tumors formed when 103 cells were injected into the mammary fat pad of NOD/SCID mice. Conclusion The CMSs isolated from tumor tissues are rich in breast cancer stem cells and similar to the mammospheres obtained by the suspension culture. It indicats that the isolation of the microspheres in breast cancer stem cells after neoadjuvant chemotherapy may be a new method to obtain breast cancer stem cells.

Abstract:Objective To establish the method of high performance liquid chromatography (HPLC) for determining quercetin and kaempferol in Qianliebeixi tablet,and provide basis for the quality control of industrial production. Methods The method of HPLC was developed to determine quercetin and kaempferol in Qianliebeixi tablet. The developed conditions of HPLC were as follows:the shim-pack     VP-ODS C18(150.0 mm×4.6 mm,5 μm),Acetonitrile-0.2% phosphoric acid solution(30∶70) mobile phase,370 nm detection wave length,and 30℃ column temperature. Results The linear range of quercetin was 0.042 6-0.639 0 μg, the regression equation was Y=－10 931+1 426 905X，r=0.999 8, the average recovery was 98.5%,and the RSD was 2.61% (n=6). The linear range of kaempferol was 0.045 4～0.726 4 μg,the regression equation was Y=－55 868+1 656 625X，r=0.999 2, the average recovery was 100.3%,and the RSD was 2.28% (n=6). The RSD in accuracy,stability and repeatability test were 1.66%,2.11%,and 1.35%,respectively. The content of quercetin or kaempferol in Qianliebeixi tablet was limited not less than 0.7 mg per one tablet under the developed condition. Conclusion The method of HPLC for determining quercetin and kaempferol in Qianliebeixi tablet is accurate，simple and reliable,which provides the basis for the quality control of Qianliebeixi tablet.

Abstract:Objective To establish  the method for detection of biological activity of basic fibroblast growth factor(bFGF )protein sponge in vitro and investigate the stability of bFGF protein sponge stored  at room temperature.Methods The bFGF biological protein sponge was fused into the dilution of the test solution,the extract was obtained after centrifugation,the bFGF standard samples were used as   control,and the bFGF protein sponge was cultivated with NIH3T3 cells.MTT assay was used to dected the proflieration  of NIH3T3 cells induced with bFGF biological protein sponge extract.The   bFGF protein  sponge stored for 2,3,4 years at room temperature was used as  the test materials,the bFGF biological protein sponge stored for 30 d at low temperature  as  standard  control,the culture medium without  bFGF protein  sponge and  bFGF  as blank control,the  potency of biological activity of each group was detected.The   stability of biological activities in vitro  of  bFGF protein sponge stored for different time were compared.Results Compared with blank  control,the A value of bFGF biological protein sponge extract was increased with the increasing of concentration,in other words the  NIH3T3 cell proliferation was increased(P<0.01);compared with bFGF standard control, the difference was not significant (P> 0.05),either bFGF standard or bFGF biological protein sponge showed a dose-response relationship  by the developed method,and the determination result complied with the following four-parameter equation:y =（A-D）／［1 +（X ／ C）B］+D. Compared with protein sponge stored for 30 d at low temperature,the descending rate of biological activity titers (U?mL-1) of the protein sponge stored for 2 to 4 years at room temperature  were  0.5%,0.6% and 0.8%,lower than the national standard of 15%. Conclusion The NIH3T3 cell proliferation could be applied for detection of the biological activity of bFGF protein sponge in vitro,it could be used as the routine detection method for bFGF protein sponge,the stabilities of bFGF protein sponge stored for 2-4 years at room temperature are good.

Abstract:Objective To establish a rapid,specific and sensitive method for the detection of Helicobacter pylori,and provide an effective detecting evidence for Helicobacter pylori. Methods A pair of primers and probe correspnding to the urease gene for real time PCR were designed according to Primer Express 3.0 software,similar sequences were searched by Blast method,and the excellent primers and probe were selected. DNA from common bacterial pathogens such as Escherichia coli,Staphylococcus,Listeria monocytogenes,Campylobacter and Salmonella in food was used for specific test;the counted Helicobacter pylori in bacterium suspension and the bacterium and milk mixture were serially diluted,the DNA was extracted for  real time PCR amplification,the sensitivity of method was tested. The practicability of the method was demonstrated through the detection of the artificial contaminative samples by real time PCR. Results The primers and probe according to urease gene could only amplify Helicobacter pylori DNA,but not  other reference bacterium DNA. Helicobacter pylori from the artificial contaminative samples could be amplified by the method. It can detect 3 CFU/mL of Helicobacter pylori. Its sensitivity was sufficient to detect 3 CFU/mL of Helicobacter pylori. The method was enough rapid to finish detection in 4 d.Conclusion Real time PCR can rapidly and accurately detect the Helicobacter pylori contamination in food with high specificity and sensitivity.