miR-133a-3p重组腺相关病毒的构建及抑制LX-2细胞α-SMA基因表达  

Construction of AAV8 that over -expresses miR-133a-3p and its inhibitory effect on α-SMA expression in LX-2 cells

作　　者：李亮 张传山 毕晓娟 马洋洋 王慧 吐尔干艾力·阿吉 林仁勇 LI Liang;ZHANG Chuan-shan;BI Xiao-juan;MA Yang-yang;WANG Hui;TUERGANAILI Aji;LIN Ren-yong(State Key Laboratory of Pathogenesis, Prevention and Treatment of Highly Prevalent Diseases in Central Asia, Clinical Medical Research Institute, The First Hospital Affiliated with Xinjiang Medical University,Urumqi, China 830054;Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University;Department of Biochemistry, College of Basic Medicine , Xinjiang Medical University)

机构地区：[1]省部共建中亚高发病成因与防治国家重点实验室,新疆医科大学第一附属医院临床医学研究院,新疆乌鲁木齐830054 [2]新疆大学生命科学与技术学院,新疆生物资源基因工程重点实验室 [3]新疆医科大学基础医学院生物化学与分子生物学教研室

出　　处：《中国病原生物学杂志》2019年第6期625-628,共4页Journal of Pathogen Biology

基　　金：新疆维吾尔自治区自然科学基金项目(No.2016D01C329);国家自然科学基金项目(No.81371838,81660108);新疆维吾尔自治区创新环境(人才、基地)项目(No.201705120)

摘　　要：目的构建miR-133a-3p重组腺相关病毒(recombinant adeno-associated virus, rAAV)过表达载体并转染肝星状细胞,鉴定其转染及干预作用。方法体外培养肝星状细胞株LX-2细胞,试验分为DMEM对照组,AAV8-Scrambled对照组,AAV8-miR-133a-3p组,应用qRT-PCR法检测miR-133a-3p转染表达量,检测胶原蛋白(COL1A1)、-平滑肌肌动蛋白(alpha smooth muscle actin,α-SMA)和TGF-β/Smad信号通路基因的表达量。结果重组腺相关病毒成功构建并转染LX-2细胞,AAV8-miR-133a-3p转染LX-2细胞组的miR-133a-3p相对表达量为11 000±343.10,与DMEM对照组比较差异有统计学意义(t=32.07,P<0.01)。AAV8-miR-133a-3p转染组与DMEM组α-SMA mRNA的相对表达量分别为0.42±0.06 vs 1.00±0.09(t=5.72,P<0.05),COL1A1 mRNA的相对表达量为0.34±0.03 vs 1.00±0.04(t=12.66,P<0.01)。结论重组腺相关病毒成功转染LX-2细胞,且能抑制肝星状细胞纤维化标志物α-SMA的表达,为探讨miR-133a-3p在调控肝星状细胞所致纤维化中的作用奠定了理论基础。Objectives To construct an AAV vector that over-expresses miR-133 a-3 p and to examine its inhibitory effect on α-SMA mRNA expression in the LX-2 hepatic stellate cell line. Methods LX-2 cells were cultured in vitro, divided into three groups, and transfected with three agents(DMEM control, AAV8-Scrambled group and AAV8-miR-133 a-3 p group). The relative expression of miR-133 a-3 p and the expression of α-SMA, COL1 A1, caspase 9, and TGF-β/Smad mRNA were detected using qRT-PCR. Results AAV8-miR-133 a-3 p was successfully constructed and transfected into LX-2 cells. miR-133 a-3 p was over-expressed in the AAV8-miR-133 a-3 p group. The relative level of expression of α-SMA mRNA in LX-2 cells transfected with AAV8-miR-133 a-3 p was 0.42±0.06. The level differed significantly from that in the DMEM control group(t=5.72, P<0.05). The relative level of expression of COL1 A1 mRNA was 0.34±0.03. The level differed significantly from that in the DMEM control group(t=12.66, P<0.01). There were no significant differences in expression of TGF-β/Smad mRNA(P>0.05). Conclusion AAV8-miR-133 a-3 p was successfully constructed and transfected into the LX-2 cell line. The results of inhibition of over-expression of miR-133 a-3 p indicated that AAV8-miR-133 a-3 p is a novel tool for further research on liver fibrosis regulated by HSC and miR-133 a-3 p.

关 键 词：腺相关病毒 肝星状细胞 miR-133a-3p 

分 类 号：R373.9[医药卫生—病原生物学]