Daxx过表达能显著抑制AngⅡ诱导的血管平滑肌细胞的增殖和迁移  

Daxx overexpression inhibits AngⅡ-induced proliferation and migration in vascular smooth muscle cells

作　　者：曹玉梅 孙四玉 杨冬梅[2] 霍艳杰 邱飞 谢雪娇 庹勤慧[1,2] CAO Yumei;SUN SiYu;YANG Dongmei;HUO Yanjie;QIU Fei;XIE Xuejiao;TUO Qinhui(School of Pharmacy,,Hunan University of Chinese Medicine,Changsha 410208,China;School of Medicine,Hunan University of Chinese Medicine,Changsha 410208,China)

机构地区：[1]湖南中医药大学药学院,湖南长沙410208 [2]湖南中医药大学医学院,湖南长沙410208

出　　处：《南方医科大学学报》2019年第10期1173-1179,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(81673722,81603600);湖南省教育厅重点项目(16A156);湖南省自然科学基金(2019JJ50426);湖南中医药大学基础医学一流学科开放基金(2018JCYX04);校级研究生科研创新项目(2018CX54)~~

摘　　要：目的构建重组慢病毒表达载体pCDH-Daxx-EGFP,并探讨Daxx对血管紧张素Ⅱ(AngⅡ)诱导VSMCs增殖的影响。方法基于PAS(PCR-based Accurate Synthesis)的方法构建重组慢病毒表达载体pCDH-Daxx-EGFP。经测序和酶切验证后,用重组慢病毒表达载体pCDH-Daxx-EGFP与包装辅助载体共转染293T细胞进行慢病毒包装,收集病毒液纯化后感染VSMCs,Western blot法鉴定过表达Daxx的VSMCs株。然后将pCDH-EGFP病毒液感染的空载体细胞和pCDH-Daxx-EGFP病毒液的感染的过表达Daxx细胞都分成无血清培养基孵育组和AngⅡ孵育组。用MTT法检测细胞活性、流式细胞术观察细胞周期、划痕法检测细胞迁移、Western blot法检测细胞中p-Akt蛋白表达。结果基因测序与双酶切鉴定表明Daxx基因慢病毒表达载体构建成功;与Vector组比,转染Daxx组、蛋白表达水平明显增加(P<0.05);经AngII处理后,转染Daxx组细胞活性、S期细胞比率和与细胞迁移率与Vector组比较显著降低(P<0.05);转染Daxx组细胞中p-Akt蛋白表达显著降低(P<0.05)。结论成功构建了重组慢病毒表达载体pCDH-Daxx-EGFP和过表达Daxx的VSMCs株;Daxx能显著抑制AngII诱导的VSMCs增殖和迁移,其机制可能与p-Akt蛋白有关。Objective To construct a recombinant lentiviral expression vector pCDH-Daxx-EGFP to investigate the effect of Daxx on the proliferation of vascular smooth muscle cells(VSMCs).Methods The recombinant lentiviral expression vector pCDHDaxx-EGFP was constructed using PCR-based accurate synthesis method.After identification by sequencing and enzyme digestion,the recombinant lentiviral vector was contransfected into 293T cells with lentivirus packaging vector.The recombinant lentivirus particles were collected and purified to infect VSMCs,whose expression of Daxx was detected with Western boltting.The cells infected with the empty vector pCDH-EGFP or pCDH-Daxx-EGFP were incubated in serum-free medium or in the presence of angiotensin II(AngII).The cell viability was determined with MTT assay,and the cell cycle changes were analyzed with flow cytometry.The cell migration ability was assessed using a scratch wound healing assay.The expression of p-Akt protein in the cells was detected using Western blotting.Results Double enzyme digestion and sequencing confirmed successful construction of the recombinant plasmid.Compared with the cells infected with the empty vector,the cells infected with pCDH-Daxx-EGFP exhibited significantly increased expressions of Daxx protein(P<0.05).AngII treatment of the cells infected with the pCDH-Daxx-EGFP,as compared with the cells infected with the empty vector,significantly lowered the cell viability,S phase cell ratio and cell migration ability(P<0.05),and significantly decreased the expression level of p-Akt protein(P<0.05).Conclusion We successfully constructed the recombinant lentiviral vector pCDH-Daxx-EGFP and overexpressed Daxx in primary cultured VSMCs using this vector.Daxx overexpression can inhibit AngII-induced proliferation and migration in VSMCs probably by regulating p-Akt protein.

关 键 词：DAXX 慢病毒载体 血管平滑肌细胞 增殖 

分 类 号：R73[医药卫生—肿瘤]