稳定高表达MT-1E基因人肾小管上皮HK-2细胞株的构建  

Construction of human renal tubular epithelial HK-2 cell line stably over-expressing MT-1E gene

作　　者：李莎[1] 罗磊[1] 黄邵玲[1] 李继猛[1] 熊艳艳 朱雪芹 谢颖[2] LI Sha;LUO Lei;HUANG Shao-ling;LI Ji-meng;XIONG Yan-yan;ZHU Xue-qin;XIE Ying(Changsha Center for Diseases Prevention and Control,Changsha 410004,Hunan,China;不详)

机构地区：[1]长沙市疾病预防控制中心,湖南长沙410004 [2]湖南师范大学医学院分子流行病学湖南省重点实验室,湖南长沙410081

出　　处：《广东医学》2022年第4期416-421,共6页Guangdong Medical Journal

基　　金：湖南省自然科学基金项目(2021JJ30752);湖南省卫生健康委科研项目(202112011277)。

摘　　要：目的成功构建高表达金属硫蛋白MT-1E基因的人肾小管上皮HK-2细胞株。方法利用转染技术将测序鉴定后的质粒转染至HK-2细胞,使用嘌呤霉素筛选转染的HK-2细胞,荧光显微镜观察并记录,通过western blot技术对阴性对照细胞组和高表达MT-1E组进行MT-1E蛋白表达量鉴定。结果转染后的质粒测序结果包含MT-1E基因序列,嘌呤霉素筛选最小致死浓度为4μg/mL,高表达MT-1E组蛋白相对表达量为2.79±0.16,与阴性对照细胞组比较,差异有统计学意义(t=15.89,P=0.016)。结论本研究完成高表达MT-1E基因的HK-2细胞株构建,可用于后续相关分子生物学实验。Objective Constructing HK-2 renal tubular epithelial cell lines with over-expression of metallothionein(MT)-1 E gene.Methods Plasmid DNA was conducted and identified by sequencing,and then was expressed in HK-2 cells.After selected by puromycin,transfection cells were observed by fluorescence microscopy.And quantitative analysis of negative control cell group and over expression group MT-1 E protein performed using western blot.Results The successful plasmid construction was verified by DNA sequencing.The minimum lethal concentration of puromycin was 4μg/mL,and the relative MT-1 E protein expression in plasmid transfection group was 2.79±0.16,which was significant higher compared with that in control group(t=15.89,P=0.016).Conclusion HE-2 cell lines stably overexpressed MT-1 E are constructed,which can be used for the subsequent related molecular biology experiments.

关 键 词：人肾小管上皮细胞 金属硫蛋白 质粒转染 

分 类 号：R134.1[医药卫生—劳动卫生] R114[医药卫生—公共卫生与预防医学]