作　　者： ; ; ; ; ;

机构地区： 深圳市疾病预防控制中心

出　　处： 《中华劳动卫生职业病杂志》 2019年第1期13-19,共7页

摘　　要： 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(Westernblot)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24h,检测凋亡基因Bax、Caspase-3、Caspase-8的基因和蛋白表达水平的变化。结果该实验插入慢病毒载体PLVX-shRNA2-puro中的3β-HSD基因干扰序列与设计的序列一致;3β-HSD沉默细胞中3β-HSD基因表达水平较对照组MCF-7细胞降低77%,3β-HSD蛋白表达水平较对照组MCF-7细胞降低74%。不同剂量DEHP染毒3β-HSD沉默细胞和MCF-7细胞后,qRT-PCR结果显示,MCF-7细胞中Bax基因表达水平较对照组(0mmol/LDEHP)升高28%~54%,Caspase-3基因表达水平升高13%~49%,Caspase-8基因表达升高21%~70%。与同染毒剂量组的MCF-7细胞比较,3β-HSD沉默细胞中Bax基因表达水平降低11%~28%,Caspase-3基因表达降低12%~23%,Caspase-8基因表达降低11%~34%。Westernblot结果显示,MCF-7细胞中BAX蛋白表达水平较对照组升高28%~61%,Caspase-3蛋白表达升高40%~48%,Caspase-8蛋白表达升高31%~84%。与同染毒剂量组的MCF-7细胞比较,3β-HSD沉默细胞Bax蛋白表达水平降低11%~27%,Caspase-3蛋白表达降低21%~40%,Caspase-8蛋白表达降低12%~25%。结论该研究成功构建了3β-HSD基因沉默细胞株。DEHP可引起细胞凋亡基因及蛋白表达升高,3β-HSD基因沉默能在一定程度上抑制DEHP对凋亡基因的活化。 Objective To construct 3β-HSD gene shRNA lentivirus interference vecto, then transfect into human MCF-7 cells, and construct cell line with 3β-HSD gene silencing, finally to study the effects of 3β-HSD on apoptosis induced by di-(2-ethylhexyl) phthalate (DEHP). Methods According to the mRNA sequence of 3β-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 screening, MCF-7 cells with 3β-HSD gene silencing were constructed. The cells with 3β-HSD gene silencing were identified by real-time quantitative PCR and western blot. Then the 3β-HSD gene silencing cells and MCF-7 cells were treated at various doses of DEHP for 24 hours to detect the gene expression and protein expression of apoptosis genes including Bax, Caspase-3 and Caspase-8. Results The interference sequence of 3β-HSD gene inserted into lentivirus vector PLVX-shRNA2-puro is consistent with the designed sequence. 3β-HSD gene expression level in MCF-7 cells with 3β-HSD gene silencing was 77% lower than than that of control MCF-7 cells. 3β-HSD protein level in MCF-7 cells with 3β-HSD gene silencing was 74% lower than that of control MCF-7 cells. After DEHP treatment in MCF-7 cells with 3β-HSD gene silencing and control MCF-7 cells, qRT-PCR results showed that Bax gene expression levels increased by 28%-54%, Caspase-3 gene increased by 13%-49%, Caspase-8 gene increased by 21%-70% in MCF-7 cells when compared with the control group. Additionally, in the 3β-HSD gene silencing cells, Bax gene expression level decreased by 11%-28%, Caspase-3 gene expression decreased by 12%-23%, Caspase-8 gene expression decreased by 11%-34%, compared with the same treatment group of MCF-7 cells. Western blot results showed that Bax protein expression level increased by 28%-61%, Caspase-3 protein expression level increased by 40%-48%, Caspase-8

关 键 词： 羟类固醇脱氢酶 基因沉默 邻苯二甲酸二 乙基己 酯 细胞凋亡

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