| Objectives To investigate the effects of SH2 domain-containing protein tyrosine phosphatase 2(SHP2)on the cell cycle of activated hepatic stellate cells(HSC)in vitro.Methods By repeatedly infecting the AD293 cells,the control empty virus Ad-GFP only expressing the green fluorescent protein(GFP),the recombinant adenovirus Ad-SHP2 carrying the wild-type SHP2(PTPN11)gene and expressing GFP and the recombinant adenovirus Ad-shRNA/SHP2 carrying the SHP2-targeting RNA interference sequence--short hairpin RNA(shRNA)and expressing GFP were amplified.The human hepatic stellate cell line LX-2 was cultured in vitro,and the recombinant adenovirus Ad-SHP2,Ad-shRNA/SHP2,and the empty virus Ad-GFP were transfected into HSC.The cell cycle phase of HSC in each group was detected by flow cytometry,and Western blot and Realtime PCR techniques were used to detect the expression changes of SHP2,Cyclin D1,CDK4 and p27kip1 of HSC in each group.Experimental groups:(1)Control group:DMEM was used instead of virus for adenovirus transfection.(2)Ad-GFP group: HSC were transfected with empty virus.(3)Ad-shRNA/SHP2 group: HSC were transfected with recombinant adenovirus Ad-shRNA/SHP2.(4)Ad-SHP2 Group: HSC were transfected with recombinant adenovirus Ad-SHP2.Results 1 After AD293 cells were repeatedly infected with adenovirus,the adenovirus with the titer required for the experiment was obtained.Adenovirus Ad-SHP2,AdshRNA/SHP2 and AD-GFP were transfected into HSC,and the transfection efficiency of adenovirus all reached more than 80% within 48 hours.2 The HSC infected with adenovirus for 48 hours were collected and the expression of SHP2 mRNA and protein in each group was detected by real-time quantitative PCR and Western blot.The expression of SHP2 mRNA and protein of HSC in the Ad-SHP2 group were significantly higher than those in the Control group,Ad-GFP group,and AdshRNA/SHP2 group(P<0.05).The expression of SHP2 mRNA and protein of HSC in the Ad-shRNA/SHP2 group were significantly lower than those in the Control group,Ad-GFP group and Ad-SHP2 group(P<0.05).However,there was no significant difference in the expression of SHP2 mRNA and protein of HSC between the Control group and the AdGFP group(P>0.05).3 The cell cycle phase of HSC in each group detected by flow cytometry showed that the percentage of cells in G0/G1 phase in the Ad-SHP2 group(43.908%±1.575%)was significantly lower than that in the Control group(52.189%±0.518%),Ad-GFP group(52.145%±0.808%)and Ad-shRNA/SHP2 group(72.933%±1.026%,P<0.05),while the percentage of cells in Ad-shRNA/SHP2 group was significantly higher than those in the Control group,Ad-GFP group and Ad-SHP2 group(P<0.05).The percentage of S-phase cells in the Ad-SHP2 group(32.068%±1.706%)were significantly higher than those in the Control group(29.490%±1.775%),Ad-GFP group(29.167%±1.225%)and Ad-shRNA/SHP2 group(13.443%±1.769%,P<0.05).However,the percentage of Ad-shRNA/SHP2 group was significantly lower than those in the Control group,Ad-GFP group and Ad-SHP2 group(P<0.05).The percentage of cells in G2/M phase in the Ad-SHP2 group(24.023%±2.326%)was significantly higher than those in the Control group(18.321%±2.008%),Ad-GFP group(18.688%±1.980%)and AdshRNA/SHP2 group(13.623%±2.272%,P<0.05).However,the percentage in the AdshRNA/SHP2 group was significantly lower than those in the Control group,Ad-GFP group and Ad-SHP2 group(P<0.05).In addition,there was no significant difference in the percentage of cells in each cell cycle phase between the Control group and the Ad-GFP group(P>0.05).4 When HSC were infected with adenovirus for 48 hours,the expressions of Cyclin D1 and CDK4 mRNA and protein of HSC in each group were detected by realtime quantitative PCR and Western blot.The expression of Cyclin D1 and CDK4 mRNA and protein of HSC in Ad-SHP2 group were significantly higher than those in the Control group,Ad-GFP group and Ad-shRNA/SHP2 group(P<0.05).However,the expressions of Cyclin D1 and CDK4 mRNA and protein of HSC in the Ad-shRNA/SHP2 group were significantly lower than those in the Control group,Ad-GFP group,and Ad-SHP2 group(P<0.05).But there was no significant difference in the expressions of Cyclin D1 and CDK4 mRNA and protein of HSC between the Control group and Ad-GFP group(P>0.05).5 When HSC were infected with adenovirus for 48 hours,the expressions of p27kip1 mRNA and protein of HSC in each group were detected by real-time quantitative PCR and Western blot.The expressions of p27kip1 mRNA and protein of HSC in the Ad-SHP2 group were significantly lower than those in the Control group,Ad-GFP group,and AdshRNA/SHP2 group(P<0.05).However,the expressions of p27kip1 mRNA and protein of HSC in Ad-shRNA/SHP2 group were significantly higher than those in the Control group,Ad-GFP group and Ad-SHP2 group(P<0.05).But there was no significant difference in the expressions of p27kip1 mRNA and protein of HSC between the Control group and the Ad-GFP group(P>0.05).Conclusions 1 The up-regulation of SHP2 expression promotes the transition of the cell cycle of G1-S phase in activated HSC in vitro,while the down-regulation of SHP2 expression significantly inhibits the transition of the cell cycle of G1-S phase in activated HSC in vitro,and blocks the cell cycle of HSC in the G0-G1 phase.2 The expression changes of Cyclin D1,CDK4 and p27kip1 are involved in the regulation of the cell cycle of activated HSC in vitro by SHP2.That is to say,up-regulation of SHP2 expression promotes the transition of the cell cycle of G1-S phase through the increase of Cyclin D1 and CDK4 expressions and the decrease of p27kip1 expression in activated HSC in vitro,while down-regulation of SHP2 expression inhibits the transition of the cell cycle of G1-S phase by the decrease of Cyclin D1 and CDK4 expressions of activated HSCs in vitro,as well as the increase of p27kip1 expression.Figure 6;Table 13;Reference 106... |
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