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

摘要:

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：①Control组，以DMEM代替腺病毒转染LX-2细胞；②Ad-GFP组，转染空病毒Ad-GFP；③Ad-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。

Abstract:

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 (qRT-PCR) 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．