Objective: To investigate the effect of adenovirus-mediated short hairpin RNA (shRNA) downregulating SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) on the apoptosis of human hepatic stellate cells LX-2 cultured in vitro. Methods: The recombinant adenovirus Ad-shRNA/SHP2 carrying shRNA targeted SHP2 and expressing green fluorescent protein (GFP), and the empty control virus Ad-GFP expressing GFP were transfected into LX-2 cells cultured in vitro. Real-time fluorescence quantitative PCR was used to detect SHP2 mRNA expression in LX-2 cells. Western blot was used to detect the protein expressions of SHP2, Bax, and Bcl-2 in LX-2 cells. TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry were used to detect apoptosis in LX-2 cells. Experimental group: (1) Control group: LX-2 cells were transfected with DMEM instead of adenovirus; (2) Ad-GFP group: transfected with empty virus Ad-GFP; (3) Ad-shRNA/SHP2 group: transfected with recombinant adenovirus Ad-shRNA/SHP2. The means between multiple groups were compared using a one-way ANOVA and the LSD test was used for inter group comparisons. Results: shRNA-targeted SHP2 significantly down-regulated the expression of SHP2 protein and mRNA in LX-2 cells (P < 0.05). The TUNEL and annexin-V/propidium iodide dual-labeled flow cytometry results showed that the apoptosis rate of LX-2 cells in the Ad-shRNA/SHP2 group (12.755%±1.606%, 19.340%±2.505%) (P < 0.05) was significantly higher compared to the control group (3.077%±0.731%, 9.438%±0.804%) and the Ad-GFP group (3.250%±0.851%, 8.893%±1.982%), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Western blot analysis of Bax and Bcl-2 protein expression in LX-2 cells of each group revealed that the Bax protein expression was significantly higher in the Ad shRNA/SHP2 group (2.493 ± 0.203) (P < 0.05) compared to the control group and Ad-GFP group (1.989 ± 0.147, 1.999 ± 0.162), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05), while the Bcl-2 protein was significantly decreased in the Ad-shRNA/SHP2 group (1.042±0.148) compared with the control group and the Ad-GFP group (1.707±0.146, 1.521±0.142), with no statistically significant difference between the control group and the Ad-GFP group (P > 0.05). Conclusions: SHP2 expression down-regulation induces apoptosis of human hepatic stellate cells LX-2 in vitro by reducing Bcl-2/Bax.
目的： 探讨腺病毒介导的短发夹RNA (shRNA)下调含SH2结构域的蛋白酪氨酸磷酸酶2（SHP2）对体外培养的人肝星状细胞LX-2凋亡的影响。 方法： 将携带靶向SHP2的shRNA并表达绿色荧光蛋白(GFP)的重组腺病毒Ad-shRNA/SHP2及仅表达GFP的对照空病毒Ad-GFP分别转染体外培养的LX-2细胞；实时荧光定量PCR检测LX-2细胞的SHP2 mRNA表达；用Western blot检测LX-2细胞的SHP2、Bax及Bcl-2蛋白表达；TUNEL及膜联蛋白-V/碘化丙啶双标记流式细胞术检测LX-2细胞凋亡。实验分组：（1）对照组：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-shRNA/SHP2组：转染重组腺病毒Ad-shRNA/SHP2。多组间均数比较用单因素方差分析，组间比较采用LSD检验。 结果： 靶向SHP2的shRNA显著下调LX-2细胞的SHP2蛋白及mRNA表达(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，与对照组LX-2细胞凋亡率(3.077%±0.731%，9.438%±0.804%)及Ad-GFP组(3.250%±0.851%，8.893%±1.982%)比较，Ad-shRNA/SHP2组LX-2细胞凋亡率(12.755%±1.606%，19.340%±2.505%)明显升高(P < 0.05)，而对照组与Ad-GFP组之间差异无统计学意义(P > 0.05)。Western blot检测各组LX-2细胞的Bax及Bcl-2蛋白表达结果显示，与对照组及Ad-GFP组LX-2细胞的Bax蛋白表达(1.989±0.147，1.999±0.162)比较，Ad-shRNA/SHP2组LX-2细胞Bax蛋白表达(2.493±0.203)显著升高（P < 0.05），而对照组与Ad-GFP组之间差异无统计学意义(P > 0.05)；与对照组及Ad-GFP组LX-2细胞的Bcl-2蛋白表达(1.707±0.146，1.521±0.142)比较，Ad-shRNA/SHP2组LX-2细胞Bcl-2蛋白表达(1.042±0.148)明显降低(P < 0.05)，而对照组与Ad-GFP组之间差异无统计学意义(P > 0.05)。 结论： SHP2表达下调通过降低Bcl-2/Bax诱导体外人肝星状细胞LX-2凋亡。.
Keywords: Apoptosis; Hepatic stellate cells; SHP2.