利用CRISPR/Cas9技术抑制Gp96的表达对小鼠酒精性肝纤维化过程中α-SMA和TGF-β1表达的影响  

Targeting Gp96 by CRISPR/Cas9 to reflect on the expression ofα-SMA and TGF-β1 in alcohol induced liver fibrosis of mice

作　　者：朱文枫 李三强[1] 宋晓改 郭威 杨欢 张兵兵 王玉东 代新华 袁旭静 朱永基 ZHU Wenfeng;LI Sanqiang;SONG Xiaogai;GUO Wei;YANG Huan;ZHANG Bingbing;WANG Yudong;DAI Xinhua;YUAN Xujing;ZHU Yongji(Key Laboratory of Molecular Medicine for Liver Injury and Repair,Medical College,He’nan University of Science and Technology,Luoyang 471000,China)

机构地区：[1]河南科技大学医学院肝脏损伤与修复分子医学重点实验室河南省肝病防治工程技术研究中心,河南洛阳471000

出　　处：《胃肠病学和肝病学杂志》2020年第11期1279-1282,1286,共5页Chinese Journal of Gastroenterology and Hepatology

基　　金：国家自然基金资助项目(81201558);河南省科技创新杰出青年(184100510006);河南省高校科技创新团队项目(18IRTSTHN026)。

摘　　要：目的研究小鼠酒精性肝纤维化进程中,利用CRISPR/Cas9技术抑制Gp96的表达对小鼠肝脏中α-SMA和TGF-β1表达的影响。方法健康雄性C57BL/6J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),尾静脉注射质粒Gp96-sgRNA3+酒精诱导肝纤维化组(n=70),腹腔注射NF-κB抑制剂PDTC+酒精诱导肝纤维化组(n=70)。于酒精诱导第8周眼球取血处死各组小鼠,测定血清谷丙转氨酶(ALT);天狼猩红染色检测各组胶原纤维变化;免疫印迹(Western blotting)检测α-SMA和TGF-β1表达变化。结果与正常对照组相比,其余三组ALT显著上升(P<0.01),胶原纤维显著增加(P<0.01),α-SMA和TGF-β1表达显著升高(P<0.01),肝纤维化加重;与生理盐水+酒精诱导肝纤维化组相比,尾静脉注射质粒Gp96-sgRNA3+酒精诱导肝纤维化组和腹腔注射NF-κB抑制剂PDTC+酒精诱导肝维化组的ALT上升更为明显(P<0.01),胶原纤维增加更显著(P<0.01),α-SMA和TGF-β1表达提升更显著(P<0.01),提示肝纤维化加剧更显著。结论小鼠酒精性肝纤维化进程中,Gp96可能通过下调α-SMA和TGF-β1的表达来减轻肝纤维化程度,而NF-κB信号通路通过上调Gp96的表达进而抑制α-SMA和TGF-β1的表达,起到减轻肝纤维化的作用。Objective To investigate the course of alcohol induced liver fibrosis in mouse,the influence of targeting protein Gp96 on the expressions ofα-SMA and TGF-β1 in the whole process.Methods 220 male healthy C57BL/6J mouse were provided into four groups randomly:normal group(n=10),merely liver fibrosis group(n=70),Gp96-sgRNA3 group(n=70),inhibitor PDTC of NF-κB signal pathway group(n=70).All black mice were killed by taking blood from their eyeballs at the last 8 week.Serum was separated to detect the activity of ALT;Hepatocyte collagen stain was taken to measure the changes of Hepatocyte collagen.The expressions ofα-SMA and TGF-β1 were detected by Western blotting.Results Compared with normal group,the activity of ALT was significantly increased in three trial groups(P<0.01),the level of ALT was significantly increased in Gp96-sgRNA3 group and NF-κB inhibition group,compared with merely liver fibrosis,liver fiber injury was induced(P<0.01).Hepatocyte collagen stain showed there were obviously liver fiber tissue in the three trial groups(P<0.01),there were even more injuried fiber liver tissues in the Gp96-sgRNA3 and NF-κB inhibition groups,compared with the merely liver fibrosis group(P<0.01).Western blotting showedα-SMA and TGF-β1 were weak and low expressions in the merely alcohol injected group(P<0.01),α-SMA and TGF-β1 were the most obvious and highest expressions in the Gp96-sgRNA3 group among the three trial groups(P<0.01).NF-κB inhibition PDTC group showedα-SMA and TGF-β1 were medium(P<0.01).Conclusion Gp96 plays a protective role in the whole liver fibrosis course through the inhibiting expressions ofα-SMA and TGF-β1.NF-κB may also take a protective part in the process through Gp96,inhibiting the expressions ofα-SMA and TGF-β1.

关 键 词：热休克蛋白GP96 酒精性肝纤维化 Α-SMA TGF-Β1 NF-κB信号通路 

分 类 号：R575[医药卫生—消化系统]