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Objective To detect the treatment effect of PC3 cells with triptolide on altering the expression of genes.Methods MTT assay was used to detec the inhibition of proliferation.Apoptosis was detected by Annexin-V/PI staining.RT-PCR was used to analyze the mRNA expressions of BCL-2,BAX,PIG3,P21,FAS,CASPASE3.Results Triptolide caused a time - and dose - dependent inhibition of cell proliferation,and IC50 of 24 h and 48 h were 18.3 ng/ml and 13.5 ng/ml,respectively.Compared with the 24 h group,the low concentration of triptolide(5 ng/ml,t =1.47,P ＞0.05)and the high concentration of triptolide ( 160 ng/ml,t =0.91,P ＞0.05)had no statistical significance in 48 h group,while 10 ng/ml( t =3.26,P ＜0.05),20 ng/ml( t =4.21,P ＜0.05),40 ng/ml( t =4.09,P ＜0.05),80 ng/ml( t =2.91,P ＜ 0.05 )had statistical significance.At the concentration of 18.3 ng/ml,triptolide induced PC3 cells apoptosis in a time - dependent manner.Compared with the control group,Anexin-V( + )/PI(-)was(5.42±2.21)％(t =3.52,P ＜0.05)in 6h,(13.51±3.37)％(t =6.53,P ＜0.01) 12h,(29.3 ±4.53)％ ( t =8.74,P ＜0.01) 24 h group separately,and it had statistical significance.RTPCR showed that 18.3 ng/ml triptolide up-regulated the mRNA expression of BAX and PIG3,down-regulated P21 and BCL-2.FAS and CASPASE3 did not show obvious changes.Conclusions Triptolide inhibits the proliferation of PC3 and induces apoptosis,and the changes of BCL-2,BAX,PIG3 and P21 may play an important role in the apoptosis of PC-3 cells.
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ObjectiveTo evaluate the early cellular immune responses to three kinds of hepatitis B surface antigen (HBsAg) in the immunized mice.MethodsAt day 4,the levels of IFN-γand IL-2 secreted by CD4+ and CD8+T cells which selected from splenic mononuclear cells (MNC) of the vaccinated mice were detected by enzyme-linked immunospot methods (ELISPOT) after stimulation in vitro with HBsAg MHC class Ⅰ peptide S28-39 of HBsAg or recombinant hepatitis B surface antigen(rHBsAg).ResultsAfter selected by MACs,the purity of CD3+/CD4+ and CD3+/CD8+T cell was more than 90％.The positive rate of IFN-γsecreted by CD4+T cells induced by HBsAg derived from Hansenula polymorpha(rHP) was higher than that of HBsAg derived from CHO cell (rCHO).Levels of IFN-γ secreted by CD8+T cells and IL-2 secreted by CD4+T cells induced by rHP antigen were significantly higher than those of rCHO( P＜0.05 ).Meanwhile,levels of IFN-γsecreted by CD4+T cells and CD8+T cells induced by rHP were also significantly higher than those of plasma HBsAg(pHB) (P＜0.05).ConclusionAt day 4,the cellular immune responses induced by HBsAg could be detected.But the immune responses induced by the three kinds of HBsAg are different in levels.According to early cellular immune response intensity,the rHP HBsAg are superior to the rCHO and pHB,in accordance with the high protection rate interrupting the mother-infant transmission immunized by rHP vaccine in clinical trial.It provides scientific basis for necessity of timely birth dose of HB vaccine and kind of HB vaccine for high risk newborn infants vaccinated.
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Objective To evaluate anti-HEV IgG and IgM diagnostic kits with sera from convalescent hepatitis E patients and to establish the quantification method of detecting anti-HEV lgG.Methods Detect 42 convalescent serum samples of over 6 months after onset of hepatitis E patients from Jiangsu province with anti-HEV IgM and IgG diagnostic kits. Select and mix the anti-HEV IgG positive sera which were confirmed by Western blot with ORF2 and ORF3 antigen. The mixed serum was calibrated with a WHO anti-HEV Ig standard. A series quantitative linear standard was made for quantitative detection of anti-HEV IgG in hepatitis E vaccine clinical trials phase Ⅲ. Results The positive rates of the anti-HEV IgG di-agnose kits of G, K, MP, Wantai were 71.4%, 78.6%, 92.9% and 100% respectively. The positive rates of G was lower than that of MP (χ~2 = 5.19, P<0.05) and obviously lower than Wantai (χ~2 = 11.76,P<0.01). The positive rates of K was also obviously lower than that of Wantai (χ~2 =7.96, P <0.01).The positive rates of the anti-HEV IgM diagnose kits of MP, G, X, Wantai, K were 21.4%, 7.1%,21.4%, 64.3%, 78.6% respectively. The positive rate of both K and Wantai were obviously higher than that of MP(χ~2 = 15.75 ,P<0.01 ; X2 = 27.43 ,P< 0.01). With the Western blot confirmation test, 30 and 18 sera were reactive to ORF2 and ORF3 antigen separately. The anti-HEV IgG concentration of HEV-D01 mixed by 13 samples was 57.94 U/ml by the calibration. Prepare seven 1.5-fold dilution series of quantita-tive linear standard for HEV vaccine clinical trials phase Ⅲ, concentration range from 0.077 to 0.877 U/ml. The quantitive values of high, medium and low concentrations quality control samples lay in the range of average ± 2s, and the CV of quantitative values were 16%, 16%, 12% respectively. Conclusion The quality of different anti-HEY IgM and IgG diagnose kits were different. This study had set up a set of anti-HEV IgG linear quantitative standard, which fit for detecting anti-HEV IgG antibodies quantitatively in HEVvaccine clinical trial phase Ⅲ.
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Objective To study the hepatocyte cells infected by hepatitis C virus (HCV) positive serum. Methods Human hepatocyte 7701 was incubated with HCV RNA-positive and HCV antibody(Ab) negative sera BP52. Then, the expression of HCV antigen and the presence of HCV-RNA in cell and supernatant were assayed by RT-PCR, sequence analysis, immunofluorescent staining, Western blot, confocal laser microscopy. The ultrastructural changes of infected cells were observed by electro-microscopy. Results Plus-strand RNA and minus-strand RNA were intermittently detected in cell and/or supernatant on day 7-45 after infection. Sequence analysis demonstrated that the positive DNA nucleic acids were identified with HCV 5′-non-coding region(NCR) sequence. HCV core and NS3 protein were expressed in cytoplasm of infected cells. After 2 or 3 weeks, obvious intracellular ultrastructural changes and virus-like particles were observed. Conclusion human hepatocyte 7701 could support replication of HCV in vitro, which could be a useful tool for setting up cell model of HCV infection and studying the mechanism of HCV infection.
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OBJECTIVE:To compare the in vitro antibacterial activity of azlocillin given alone with azlocillin plus tazobactam against168strains of clinically isolated pathogenic bacteria.METHODS:The antibacterial activities of two antibac_ terials against168strains of clinically isolated pathogenic bacteria in vitro were detected by two-fold dilution method.RE-SULTS:The MIC 50 and the MIC 90 of the combined therapy of azlocillin/tazobactam(4∶1)were1/32and1/64,respectively that of azlocillin given alone.CONCLUSION:The concomitant therapy of azlocillin with tazobactam improves the antibacterial activity.
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Objective The absorption and distribution of nitrogen(N),phosphorus(P),and potassium(K) of Hedyotis diffusa was studied in field tests.Methods Through regular sampling,dry matter accumulation was studied,the contents of N,P,and K were analyzed by conventional methods.Results The uptake amount of N,P,and K by H.diffusa was lower at seedling stage,but increased rapidly at branching,blossoming,and fruiting stages,and the uptake amount of N and P decreased and K was negatively absorbed at mature stage.The uptake amount of N by H.diffusa was the highest,following was K2O,while P2O5 was the lowest.In the growing season of H.diffusa,the ratio of N,P2O5,and K2O was 1.00:0.14:0.54.N Mainly distributed in leaf at seedling,branching,and blossoming stages,while P2O5 and K2O mainly distributed in stem.The mineral nutrition mostly distributed in stem,flowers,and fruits at fruiting and mature stages,N and P2O5 mostly distributed in flowers and fruits,but K2O mainly accumulated in stem.Conclusion A little fertilizer should be applied at seedling stage,oppositly the use amount of fertilizer should be increased at branching,blossoming,and fruiting stages,particularly P and K at fruiting stage.