利用CRISPR/Cas-9技术抑制热休克蛋白Gp96的表达对小鼠酒精性肝纤维化的影响

Effect of inhibition of heat shock protein Gp96 expression on alcoholic liver fibrosis in mice by CRISPR/Cas-9 technique

目的探讨热休克蛋白Gp96对小鼠酒精性肝纤维化的影响。方法健康雄性C57BL/6 J小鼠220只,随机分为4组:正常对照组(n=10),生理盐水+酒精诱导纤维化组(n=70)。尾静脉注射CRISPR表达质粒Gp96-sgRNA3+酒精诱导肝纤维化组(n=70),腹腔注射核因子κB(NF-κB)抑制剂PDTC+酒精诱导肝纤维化组(n=70)。于酒精诱导第8周眼球取血后处死各组小鼠,测定各组小鼠血清谷草转氨酶(AST)活性,HE染色检测各组小鼠肝脏病理变化,天狼猩红染色检测各组小鼠肝纤维化情况,过碘酸-雪夫(PAS)染色检测各组小鼠肝糖原变化情况;免疫印迹法检测小鼠肝脏Gp96和转化生长因子β1(TGF-β1)的表达变化。结果与正常对照组相比,其余3组AST酶活性显著上升,肝纤维化加重,糖原显著减少(P<0.01);与生理盐水+酒精组相比,注射质粒Gp96-sg RNA3+酒精组和注射NF-κB抑制剂+酒精组AST上升更为明显,肝纤维化更严重,糖原减少更多,Gp96表达显著下降,TGF-β1表达显著增加(P<0.01或P<0.05)。结论尾静脉注射CRISPR表达质粒Gp96-sgRNA3显著抑制了小鼠肝脏Gp96的表达,促进了小鼠酒精性肝纤维化程度,NF-κB信号通路对Gp96的表达起到一定的调控作用。

Objective To investigate the effects of heat shock protein Gp96 on alcoholic liver fibrosis in mice.Methods A total of 220 male healthy C57 BL/6 J mice were randomly divided into four groups:normal control group(n=10),saline+alcohol induced liver fibrosis group(n=70),the injection of CRISPR expression Gp96-sgRNA3 by tail vein+alcohol induced liver fibrosis group(n=70),the intraperitoneal injection of nuclear factor kappa B(NF-κB)inhibitors PDTC+alcohol induced liver fibrosis group(n=70).The blood was got from eyeballs and the mice were killed after 8 weeks of ethanol induction.We detected the activity of serum aspartate aminotransferase(AST)in mice of different groups.The pathological changes were detected by HE staining,sirius red staining and periodic acid-Schiff(PAS)staining in the liver of mice.The expression of Gp96 and transforming growth factorβ1(TGF-β1)were detected by Western blotting.Results Compared with the normal control group,the AST enzyme activity and liver fibrosis increased significantly,glycogen decreased significantly in other three groups(P<0.01).Compared with the saline+alcohol group,the AST enzyme activity and liver fibrosis increased more significantly,glycogen decreased more significantly,Gp96 expression decreased significantly and TGF-β1 expression increased significantly in Gp96-sgRNA3+alcohol group and NF-κB inhibitors PDTC+alcohol group(P<0.01 or P<0.05).Conclusion The injection of CRISPR expression plasmid Gp96-sgRNA3 by tail vein significantly inhibited the Gp96 expression,promoted the degree of alcoholic liver fibrosis in mice,and NF-κB signaling pathway played a certain role in regulating the expression of Gp96.