CHFR介导的蛋白PAR化参与肝纤维化DNA损伤应答的分子机制研究  

Molecular mechanism of CHFR mediated parylation of protein involved in DNA damage response to hepatic fibrosis

作　　者：蔡军[1] 刘殿刚[2] 斯琴 古艳婷 CAI Jun;LIU Dian-gang;SI Qin(Department of General Surgery,Beijing Friendship Hospital Affiliated to Capital Medical University,Beijing 101149,China;Department of General Surgery,Beijing Xuanwu Hospital,Affiliated to Capital Medical University,Beijing 100053,China;Medical Examination Center of Aviation General Hospital,Beijing 100012,China)

机构地区：[1]首都医科大学附属北京友谊医院普外科,北京101149 [2]首都医科大学宣武医院普外科,北京100053 [3]航空总医院体检中心,北京100012

出　　处：《临床和实验医学杂志》2019年第24期2577-2580,共4页Journal of Clinical and Experimental Medicine

基　　金：北京市自然科学基金(编号:19G11095)

摘　　要：目的 探讨泛素连接酶蛋白(CHFR)介导的蛋白PAR化参与肝纤维化DNA损伤应答的分子机制。方法制备和野生型C57BL/6小鼠同背景的CHFR基因敲除(CHFR-/-)小鼠构建四氯化碳肝纤维化(CCl4)模型,饲养4周后处死,通过免疫组化方法以及Western blotting方法验证小鼠肝纤维模型中DNA的损伤标记物γH2AX表达变化以及对小鼠肝星状细胞(HSC)进行激光照射诱导DNA双链断裂(DSBs)损伤,在荧光显微镜下观察CHFR和γH2AX的定位,采用sh RNA降低小鼠HSC中CHFR蛋白表达,进行放射线照射后,进行单细胞凝胶电泳(Comet)实验。结果 ①Western blotting和免疫组化结果均显示,正常肝组织中未见γH2AX,肝纤维化组织表达明显;②荧光显微镜下显示,HSC细胞核出现γH2AX与CHFR荧光表达,CHFR显著定位于DNA损伤部位,且CHFR与γH2AX表达趋势一致,同时表达于细胞核相同部位;③与野生型C57BL/6小鼠对比,CHFR-/-小鼠γH2AX阳性细胞数明显增多,放射线照射后,CHFR-/-小鼠“彗星尾巴”增加,DNA修复减弱。结论 CHFR可能通过参与DNA损伤修复过程介导HSC活化继而参与肝纤维过程。Objective To explore the molecular mechanism of checkpoint with forkhead and RING finger domains protein(CHFR)mediated parylation of protein involved in DNA damage response in liver fibrosis. Methods CHFR-/- mice with the same background as wild-type C57BL/6 mice were prepared to construct CCl 4 model. Four weeks later, immunohistochemical method and western blotting were used to verify the expression changes of DNA damage marker γH2AX in mouse liver fiber model and laser irradiation was applied to mouse hepatic stellate cell(HSC)to induce Double-strand breaks (DSBs) damage. The localization of CHFR and γH2AX was observed under fluorescence microscope. shRNA was used to reduce the expression of CHFR protein in mouse HSC. Comet experiment was carried out after radiation irradiation. Results ①Western blotting and immunohistochemistry showed that γH2AX was not found in normal liver tissues, and liver fibrosis tissues were obviously expressed. ②Fluorescence microscope showed that HSC nucleus showed fluorescence expression of γH2AX and CHFR, CHFR was significantly located at DNA damage site, and the expression trends of CHFR and γH2AX were consistent, and expressed at the same location of nucleus. ③Compared with wild-type mice, the number of γH2AX positive cells of CHFR-/- mice increased significantly. After radiation exposure, the "comet tail" of CHFR-/- mice increased and DNA repair weakened. Conclusion CHFR may mediate the activation of HSC and then participate in the liver fiber process by participating in DNA damage repair.

关 键 词：小鼠 肝纤维化 CHFR 肝星状细胞 DNA 双链断裂 

分 类 号：R73[医药卫生—肿瘤]