Study Of The Apoptosis Of Rat Hepatic Stellate Cells Induced By Rapamycin

Objective:1. To investigate the effect of different concentration Rapamycin(RAPA)on proliferation,apoptosis and the contents of collagen I of rHSCs-99 in vitro.2.To investigate the expressions of different concentration Rapamycin on Fas,P53 and Bcl-2 of rHSCs-99 in vitro.Methods:Make the rHSC-99 for four groups again: A(Control group): only administration of medium; B: RAPA low-concentration group; C: RAPA middle-concentration group; D:RAPA high-concentration group.After 48h or 72h for Rapamycin interfere in rHSC-99,we made the following expreiments:1,MTT colorimertic assay was used to detect the proliferation of rHSC-99;2,Synthesis and secretion of collagen I was detected by ELISA;3,Annexin-V FICT/PI double-label immuno fluorescence with flow cytometer was used to examine the apoptosis of rHSC-99;4,Wright-Giemsa staining method was used to observe the morphologic change of rHSC-99;5,Immunocytochemical staining was used to observe the proteinum express of Fas,P53 and Bcl-2.Results1,MTT method showed after 48h and 72h the OD of A,B,C,D four groups of rHSC-99 were 0.956±0.055,0.870±0.055,0.795±0.087,0.645±0.073 and 1.243±0.073, 1.131±0.058,1.022±0.082,0.848±0.133 respectively; Compared to control group, 100,150 nmol/L RAPA groups can inhibit rHSCs-99 proliferation(P<0.05);the survival rate of rHSCs-99 groups induced with 72h of RAPA were reduced compared with 48h groups in a time-dependent and density-dependent manner(P<0.05).2,ELISA assay showed the content of collagen I for A,B,C,D four groups were 134.350±6.133 ng/ml,136.371±7.888 ng/ml,121.901±9.943 ng/ml和114.491±8.534 ng/ml respectively; Compared to control group, 100,150 nmol/L RAPA groups can reduce the content of type I(P<0.05),RAPA significantly reduced the content of type I in a dose-dependent manner.3,Annexin-V FICT/PI double-label immuno fluorescence with flow cytometer showed the apoptosis ratio(%)of rHSCs-99 for A,B,C,D four groups were 0.3%±0.1%,4.82%±1.17%,12.97%±1.39% and 17.7%±1.39% respectively ; Compared to control group,all RAPA groups can facilitates rHSCs-99 apoptosis in a dose-dependent manner(P<0.05).4,Wright-Giemsa showed cell shrinkage, chromatin condensation and (or) ranked along inside of nuclear membrane ;5,Immunocytochemical staining showed that the number of P53 positive was increased and the Bcl-2 positive was decreased; Compared to control group,all RAPA groups have the statistical significance(P<0.05).Conclusion1,RAPA inhibits the proliferation and reduces the content of collagen I of rHSCs-99, facilitates the cell apoptosis in a dose-dependent manner;2,The mechanism of action for RAPA promote rHSCs-99 apoptosis may be through opening the Fas/Fasl apoptosis pathway and up-regulation Fas,P53 and inhibiting Bcl-2 expressing to come true.