期刊论文

Construction of 3β-HSD gene silencing cell line and its effects on apoptosis induced by DEHP

中文

中华劳动卫生职业病杂志

1001-9391

2019

37

1

13-19

10.3760/cma.j.issn.1001-9391.2019.01.003

深圳市科技研发基础研究项目

本校为第一机构

目的 构建3β-羟类固醇脱氢酶(3β-HSD)基因shRNA慢病毒干扰载体,建立3β-HSD基因沉默细胞株,研究3β-HSD基因沉默对塑化剂邻苯二甲酸二-(2-乙基己)酯(DEHP)致凋亡的影响.方法 根据GenBank提供的3β-HSD基因mRNA序列,设计合成3条干扰序列,退火后连接到PLVX-shRNA2-puro中,将重组慢病毒载体转染293FT细胞,收集病毒上清,感染MCF-7细胞.利用嘌呤霉素筛选得到3β-HSD沉默细胞株,通过实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹法(Western blot)对干扰效果进行鉴定.用不同剂量DEHP对MCF-7细胞和3β-HSD沉默细胞染毒24 h,检测凋亡基因Bax、Caspase-3、Caspase-8的基因和蛋白表达水平的变化.结果 该实验插...

Objective: To construct 3beta-HSD gene shRNA lentivirus interference vecto, then transfect into human MCF-7 cells, and construct cell line with 3beta-HSD gene silencing, finally to study the effects of 3beta-HSD on apoptosis induced by di- (2-ethylhexyl) phthalate (DEHP) . Methods: According to the mRNA sequence of 3beta-HSD gene provided by GenBank, three interference sequences were designed and connected to PLVX-shRNA2-puro after annealing. The recombinant lentivirus vector was transfected into 293FT cells, the virus supernatants were collected and infected with MCF-7 cells. After puromycin ...