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

朱文枫; 李三强; 宋晓改; 郭威; 杨欢; 张兵兵

doi:10.16098/j.issn.0529-1356.2021.05.016

解剖学报 >

2021 , Vol. 52 >Issue 5: 777 - 783

DOI: https://doi.org/10.16098/j.issn.0529-1356.2021.05.016

组织学胚胎学发育生物学

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

朱文枫 ,
李三强 ,
宋晓改 ,
郭威 ,
杨欢 ,
张兵兵

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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; 2.河南省肝病防治工程技术研究中心，河南洛阳 471000

收稿日期: 2020-02-20

修回日期: 2020-05-25

网络出版日期: 2021-10-06

基金资助

组织损伤与修复分子机制及防治研究

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

ZHU Wen-Feng ,
LI San-Qiang ,
SONG Xiao-Gai ,
GUO Wei ,
YANG Huan ,
ZHANG Bing-Bing

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1.Molecular Medicine Key Laboratory of Liver Injury and Repair, Medical College, He’nan University of Science and Technology, He’nan Luoyang 471000, China; 2.He’nan Center for Engineering and Technology Research on Prevention and Treatment of Liver Diseases, He’nan Luoyang 471000, China

Received date: 2020-02-20

Revised date: 2020-05-25

Online published: 2021-10-06

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摘要

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-sgRNA3+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。

关键词： 热休克蛋白Gp96; 酒精性肝纤维化; 规律间隔成簇短回文重复序列/相关蛋白9; 免疫印迹法; 小鼠

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朱文枫 , 李三强 , 宋晓改 , 郭威 , 杨欢 , 张兵兵 . 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[J]. 解剖学报, 2021 , 52(5) : 777 -783 . DOI: 10.16098/j.issn.0529-1356.2021.05.016

Abstract

Objective To investigate the effects of heat shock protein Gp96 on alcoholic liver fibrosis in mice. Methods A total of 220 male healthy C57BL/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 factor β1(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.

Key words： Heat shock protein Gp96; Alcohol induced liver fibrosis; Nuclear factor κB signal pathway; Clustered regularly interspaced short palindromic repeats/associated protein 9; Western blotting; Mouse

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