RNA干扰bcl-2基因的筛选及对肝星状细胞生物活性的影响

Screening of RNA interference to bcl-2 gene and the effect on biological behaviors of hepatic stellate cells

目的研究小干扰RNA（shRNA）重组载体介导抑制大鼠肝星状细胞（hepaticstellatecell，HSC）bcl-2基因的表达，初步观察其对HSC生物活性的影响。方法设计有小发夹结构的3条DNA序列构建重组质粒载体pGPU6-GFP，脂质体转染HSC—T6细胞株以荧光定量PCR和Westernblot筛选鉴定，通过CCK-8法及AnnexinV／PI双标记流式细胞术检测、观察其对HSC生长的影响。结果pGPU6-GFP—shRNA1、shRNA2均能抑制bcl-2mRNA和蛋白表达（P〈0．05），pGPU6-GFP—shRNAl转染HSC．T6株72h后对bcl-2基因抑制达80％，且HSC—T6体外生长明碌受到抑制，早期凋亡率为33．34％～44．12％。结论bel-2小发夹RNA重组载体shRNA1能最有效抑制HSC—T6中bcl-2的表达与细胞生长，促进凋亡，为下一步探索肝纤维化基因治疗提供实验依据。

Objective To investigate the effect of small hairpin RNA （shRNA） targeting bcl-2 gene on the bcl-2 expression and the biological behaviors of hepatic stellate cells （HSCs）. Methods Three designed DNA sequences with small hairpin structure were cloned into the transeripted carrier pGPU6-GFP which was transfected into HSC-T6 by lipofectamine. The expression of bcl-2 was screened by real-time PCR and Western blot. The cell proliferation was assayed by CCK-8 method and the cell apoptosis by flow cytometry. Results pGPU6-GFP-shRNA1 , shRNA2 could inhibit the expression bcl-2 mRNA and protein （P 〈 0.05） , pGPU6-GFP-shRNA1 of which was the most effective one with 80% inhibition rate, significantly decreasing the proliferating activity of HSC （ P 〈 0.05 ） , but increasing the apoptosis rate 33.34% -44.12%. Conclusion The constructed bcl-2 shRNA expressing plasmid of pGPU6-GFP-shRNA1 an reduce the expression of bcl-2 at maximum, inhibit the proliferation of HSC and induce apoptosis. Experimental basis is provided for gene therapy of liver fibrosis.