| Objectives To investigate the effects of SH2 domain-containing protein tyrosine phosphatase 2(SHP2)on the biological activity of activated hepatic stellate cells(HSC)in vitro.Methods Through repeatedly infecting AD293 cells with adenovirus,the control empty virus Ad-GFP expressing only green fluorescent protein(GFP),the recombinant adenovirus Ad-sh RNA/SHP2 expressing GFP and carrying the SHP2 targeting RNA interference sequence-short hairpin RNA(sh RNA),and the recombinant adenovirus Ad-SHP2 expressing GFP and carrying the wild-type SHP2(PTPN11)gene were amplified;The human hepatic stellate cell line LX-2 were cultured and transfected with the above adenoviruses in vitro.The cell counting kit-8(CCK8)method was applied to test the proliferation of LX-2 cells in each group.Western blot and immunocytochemistry staining were used to determine the expression of α-smooth muscle actin(α-SMA)in HSC.Realtime PCR technology was applied to test the m RNA expression changes of SHP2,ERK and Akt in HSC.Western blot was applied to test the protein expressions of SHP2,collagen type I,collagen type III,ERK1,p-ERK1,Akt and p-Akt in HSC.Experimental groups:(1)Control group,DMEM was applied instead of virus solution.(2)Ad-GFP group,Transfection with Ad-GFP.(3)Ad-sh RNA/SHP2 group,Transfection with adenovirus Adsh RNA/SHP2.(4)Ad-SHP2 group,Transfection with adenovirus Ad-SHP2.Results 1 By repeatedly infecting AD293 cells with adenovirus Ad-GFP,Adsh RNA/SHP2,and Ad-SHP2,the above three adenovirus solutions meeting the requirements of this experiment were obtained.2 HSC were infected with adenovirus for48 hours,and the adenovirus transfection efficiencies of HSC in each group were observed under an inverted fluorescence microscope,which were all greater than 80%.3 Real-time PCR and Western blot were applied to test the expression of SHP2 m RNA and protein in HSCs in each group.The expression of SHP2 m RNA and protein in HSC in Adsh RNA/SHP2 group were obviously less than that in Control group,Ad-GFP group and Ad-SHP2 group(P<0.05);while the expression of SHP2 m RNA and protein in HSC in Ad-SHP2 group were obviously more than that in Control group,Ad-GFP group and Adsh RNA/SHP2 group(P<0.05);but there was no obvious difference in SHP2 m RNA and protein expression between Control group and Ad-GFP group(P>0.05).4 The proliferation of hepatic stellate cells in each group was detected by CCK8 method.The proliferation of HSC in Ad-sh RNA/SHP2 group was obviously less than that in Control group,Ad-GFP group and Ad-SHP2 group,and the difference was obviously significant(P<0.05);The proliferation of HSC in Ad-SHP2 group was obviously more than that in Control group,Ad-GFP group and Ad-sh RNA/SHP2 group(P<0.05).While there was no statistical difference in the proliferation of HSC between Control group,Ad-GFP group(P>0.05).5 Western blot and immunocytochemistry staining were applied to detect the expression of α-SMA in HSC in each group.The expression of α-SMA in HSC in Adsh RNA/SHP2 group was obviously less than that in Control group,Ad-GFP group and AdSHP2 group,and the difference was obviously significant(P<0.05).The expression of α-SMA in HSC in Ad-SHP2 group was obviously more than that in Control group,Ad-GFP group and Ad-sh RNA/SHP2 group(P<0.05).However,there was no statistical difference in the expression of α-SMA in HSC between the Control group and the Ad-GFP group.(P>0.05).6 Western blot was applied to detect the expression of collagen type I and type III in LX-2 cells in each group.The expression of type I and type III collagen in HSC in Ad-sh RNA/SHP2 group was obviously less than that in Control group,Ad-GFP group and Ad-SHP2 group,the difference was obviously significant(P<0.05).The expression of type I and type III collagen in HSC in Ad-SHP2 group was more than that in Control group,Ad-GFP group and Ad-sh RNA/SHP2 group(P<0.05).However,there was no statistical difference in the expression of type I and type III collagen in HSC between the Control group and the Ad-GFP group(P>0.05).7 Real-time PCR and Western blot were applied to test ERK1 m RNA and protein expression in HSC in each group.There was no obvious difference in ERK1 m RNA and protein expression in HSC among four groups(P>0.05).Western blot was applied to test the expression of p-ERK1 in HSC in each group.The expression of p-ERK1 in HSC in Ad-sh RNA/SHP2 group was obviously less than that in Control group,Ad-GFP group and Ad-SHP2 group,the difference was obviously significant(P<0.05).The expression of p-ERK1 in HSC in Ad-SHP2 group was obviously more than that in Control group,Ad-GFP group and Ad-sh RNA/SHP2 group(P<0.05).There was no obvious difference in the expression of p-ERK1 in HSC between the Control group and the Ad-GFP group(P>0.05).8 Real-time PCR and Western blot techniques were applied to test Akt m RNA and protein expression in HSC in each group,and there was no obvious difference in Akt m RNA and protein expression in HSC among four groups(P>0.05).Western blot was applied to test the expression of p-Akt in HSC in each group.The expression of p-Akt in HSC in Ad-sh RNA/SHP2 group was obviously less than that in Control group,Ad-GFP group and Ad-SHP2 group,the difference was obviously significant(P<0.05).The expression of p-Akt in HSC in Ad-SHP2 group was obviously more than that in Control group,Ad-GFP group and Ad-sh RNA/SHP2 group(P<0.05).The difference in expression of p-Akt was not obviously significant in HSC between the Control group and the Ad-GFP group(P>0.05).Conclusions 1 Changes in the expression of SHP2 have significant effects on the bioactivity of activation,proliferation and collagen metabolism in activated hepatic stellate cells in vitro.That is,overexpression of SHP2 can promote the activation,proliferation and the synthesis of collagen types I and III in activated hepatic stellate cells in vitro,the down-regulation of SHP2 expression can inhibit the activation,proliferation and synthesis of type I and III collagen in activated hepatic stellate cells in vitro.2 The ERK and PI3K/Akt signaling transduction pathways may be involved in the regulation of SHP2 on the activation,proliferation and collagen metabolism of activated hepatic stellate cells in vitro.Figure 7;Table 14;Reference 68... |
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