单纯疱疹Ⅱ型microRNA-H3真核过表达载体的构建及对Hela细胞活性的影响  

Establishment of eukaryotic overexpression vector of HSV-2 microRNA-H3 and its' Role to Hela cells' activity

作　　者：何丹[1] 杨慧兰[1,2] 

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]广州军区广州总医院皮肤科,广东广州510010

出　　处：《现代预防医学》2017年第3期499-502,519,共5页Modern Preventive Medicine

基　　金：国家自然科学基金资助(编号81171511)靶向HSV-2潜伏复发的microRNA的筛选及作用研究(起止年月:(2012.01-2015.12);此研究项目获军队医疗成果二等奖(省部级);证书编号(2013-2-28-1)

摘　　要：目的人宫颈癌细胞(Hela)中表达Ⅱ型单纯疱疹病毒(HSV-2)潜伏相关转录体(LAT)编码的microRNA-H3(miR-H3),检测该mRNA对Hela细胞活性的影响。方法 PCR扩增microRNA-H3的前体序列,将其插入到pmRmcherry(cherry)载体中,并酶切测序同时验证;重组质粒转染Hela细胞,q PCR检测microRNA的相对表达水平,转染受体细胞,并用放线菌素D(Act D)诱导凋亡,MTT法、流式细胞术和Edu染色共同检测Hela细胞活性。结果双酶切、测序表明cherry/miR-H3重组质粒构建成功,q PCR检测miR-H3大量表达,MTT法、Edu染色和流式细胞术检测表明转染重组质粒cherry/miR-H3并经诱导凋亡组细胞的活性明显比空质粒转染组和未转染组细胞活性高,具有统计学意义,而与正常细胞对照组无显著差异。结论 cherry/miR-H3真核表达载体可在Hela细胞中高效表达,并具有抗Act D诱导的细胞凋亡作用。Objective To construct eukaryotic expression plasmid of microRNA - H3 encoded by latency - associated transcript of herpes simplex virus type 2（HSV - 2）, and to evaluate its effects on Hela cells＇ activity. Methods miR - H3 precursor fragment were amplified from HSV - 2 genome and cloned into a eukaryote plasmid pmR - mcherry （ cherry）, followed by restrictive endonucleases digestion and sequencing validation. Relative expression of miR - H3 was detected after actinomycin D treatment for 48h by using qPCR assay. MTT assay,flow cytometry and Edu staining were performed to determine the change of Hela activity. Results Double digestion and sequencing confirmed that the cherry/H3 was constructed successfully,and miR - H3 was expressed much in Hela cells after transfection. Data from MTT assay, flow cytometry and Edu staining demonstrated that activity of Hela ceils transfected with recombinant plasmid were much higher than untreated normal control cells and those transfected with empty plasmids, which had statistically significant difference. No significant difference was founded when contrasted with normal cells. Conclusion PmR - mcherry recombinant plasmid expressed well in Hela cells and can prevent ActD induced apoptosis which may account for miR - H3 overexpression.

关 键 词：疱疹病毒 microRNA—H3 Hela细胞 抗细胞凋亡 

分 类 号：R113[医药卫生—公共卫生与预防医学]