SHP2过表达抑制人肝星状细胞LX-2凋亡

Overexpression of SHP2 inhibits apoptosis of human hepatic stellate cells LX-2

目的探讨含SH2结构域的蛋白酪氨酸磷酸酶2(SHP2)过表达对人肝星状细胞LX-2凋亡的影响。方法将表达绿色荧光蛋白(GFP)的空病毒Ad-GFP、表达野生型SHP2及GFP的腺病毒Ad-SHP2转染LX-2细胞。实验分为3组:(1)Control组,以DMEM代替腺病毒转染LX-2细胞;(2)Ad-GFP组,转染空病毒Ad-GFP;(3)Ad-SHP2组,转染重组腺病毒Ad-SHP2。Western blotting检测3组LX-2细胞的SHP2、Bax、Bcl-2蛋白表达;实时荧光定量聚合酶链反应(qRT-PCR)检测3组LX-2细胞的SHP2 mRNA表达;TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测3组LX-2细胞凋亡情况。结果Ad-SHP2组LX-2细胞的SHP2蛋白及mRNA相对表达量高于Control组及Ad-GFP组(P<0.05);Ad-GFP组与Control组比较,差异无统计学意义(P>0.05)。TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测结果显示,与Control组LX-2细胞凋亡率(3.08±0.73)%、(9.44±0.80)%及Ad-GFP组(3.25±0.85)%、(8.89±1.98)%比较,Ad-SHP2组(1.52±0.26)%、(2.44±0.93)%降低(P<0.05);Ad-GFP组与Control组比较,差异无统计学意义(P>0.05)。3组LX-2细胞的Bax及Bcl-2蛋白相对表达量比较,差异有统计学意义(P<0.05);Ad-SHP2组LX-2细胞的Bax蛋白相对表达量(1.55±0.21)低于Control组(1.99±0.15)及Ad-GFP组(2.00±0.16)(P<0.05);而Bcl-2蛋白相对表达量(2.13±0.25)则高于Control组(1.71±0.15)及Ad-GFP组(1.52±0.14)(P<0.05);但Ad-GFP组与Control组Bax及Bcl-2蛋白相对表达量比较,差异无统计学意义(P>0.05)。结论SHP2过表达通过升高Bcl-2/Bax抑制体外人肝星状细胞LX-2凋亡。

Objective To investigate the effect of overexpression of Src homology 2 domain-containing protein tyrosine phosphatase 2(SHP2)on apoptosis in human hepatic stellate cells(LX-2).Methods LX-2 cells were transfected with adenoviruses expressing green fluorescent protein(GFP)alone(Ad-GFP),wild-type SHP2 and GFP(Ad-SHP2).The experiment was divided into three groups:Control group,in which LX-2 cells were transfected with DMEM instead of adenovirus;Ad-GFP group,transfected with empty adenovirus Ad-GFP;Ad-SHP2 group,transfected with recombinant adenovirus Ad-SHP2.Western blotting was used to detect the expression of SHP2,Bax,and Bcl-2 proteins in the three groups of LX-2 cells.Real-time quantitative polymerase chain reaction(qRTPCR)was performed to measure SHP2 mRNA expression in LX-2 cells.Apoptosis of LX-2 cells in the three groups was detected by TUNEL assay and annexin V/propidium iodide double staining flow cytometry.Results The relative expression levels of SHP2 protein and mRNA in LX-2 cells in the Ad-SHP2 group were higher than those in the Control group and Ad-GFP group(P<0.05).There was no significant difference in SHP2 expression between the Control group and Ad-GFP group(P>0.05).TUNEL assay and annexin V/propidium iodide double staining flow cytometry showed that the apoptosis rate of LX-2 cells in the Ad-SHP2 group(1.52±0.26)% and(2.44±0.93)% was lower compared to the Control group(3.08±0.73)% and(9.44±0.80)% and the Ad-GFP group(3.25±0.85)% and(8.89±1.98)%(P<0.05).There was no significant difference in apoptosis between the Control group and Ad-GFP group(P>0.05).The relative expression levels of Bax and Bcl-2 proteins in LX-2 cells in the three groups were significantly different(P<0.05).The relative expression level of Bax protein in the Ad-SHP2 group(1.55±0.21)was lower than that in the Control group(1.99±0.15)and the Ad-GFP group(2.00±0.16)(P<0.05),while the relative expression level of Bcl-2 protein(2.13±0.25)was higher than that in the Control group(1.71±0.15)and the Ad-GFP group(1.52±0.14)(P<0.05).There was no significant difference in the expression of Bax and Bcl-2 proteins between the Control group and Ad-GFP group(P>0.05).Conclusion Overexpression of SHP2 inhibits apoptosis of human hepatic stellate cells LX-2 in vitro by increasing the Bcl-2/Bax ratio.