靶向bcl-2基因的shRNA对肝星状细胞凋亡的影响

Effects of shRNA targeting bcl-2 gene on apoptosis of hepatic stellate cells

目的研究短发夹RNA(shRNA)重组载体介导抑制大鼠肝星状细胞(HSCs)bcl-2基因的表达,观察其对HSCs凋亡的影响。方法重组质粒载体pGPU6-GFP-shRNA由脂质体介导转染HSC-T6细胞株。荧光定量PCR和Western blotting鉴定bcl-2表达;CCK-8法观察质粒转染对HSCs生长和增殖的影响;Hoechst 33258染色观察细胞形态学改变;流式细胞术及caspase-3和caspase-9酶活性检测分析转染后细胞凋亡情况。结果pGPU6-GFP-shRNA能抑制bcl-2 mRNA和蛋白表达;转染后HSC-T6体外生长明显受到抑制;转染后72 h时细胞凋亡率为33.34%,caspase-3和caspase-9酶活性显著增高。结论shRNA重组载体pGPU6-GFP-shRNA能有效地抑制HSC-T6中bcl-2的表达,抑制细胞生长并促进凋亡。实验为进一步探索肝纤维化基因治疗提供了实验依据。

Objective To investigate the effects of short hairpin RNA （shRNA） targeting bcl-2 gene on bcl-2 expression and observe the apoptosis behaviors of rat hepatic stellate cells （HSCs）. Methods After lipofectamine transfection of recombinant plasmid pGPU6-GFP-shRNA expressing bcl-2 shRNA, the expression of bcl-2 was identified by real-time PCR and Western blotting. The cell proliferation was assayed by CCK-8 method,and cell morphology was observed by Hoechst 33258 staining. Apoptotic HSC-T6 cells were detected by flow cytometry and assay of activity of caspase-3 and caspase-9. Results pGPU6-GFP-shRNA inhibited the expression of bcl-2 mRNA and protein, significantly decreased the proliferating activity of HSCs, and increased the activity of caspase-3 and caspase-9. Apoptotic rate was 33.34% after transfection for 72 h. Conclusion The constructed bcl-2 shRNA expressing plasmid of pGPU6-GFP-shRNA can reduce the expression of bcl-2, inhibit the proliferation of HSCs and induce apoptosis. The experiment lays the foundation for gene therapy of liver fibrosis.