【摘要】 目的:构建特异抑制雌激素受体α(ERα)基因的短发夹RNA(sh RNA)慢病毒表达载体,检测其对ERα的干扰效果,并探讨其对MCF7细胞中乳腺癌干细胞含量的影响。方法:以人的ERα核酸序列为靶标,根据sh RNA设计原则设计并化学合成特异的sh RNA序列,经退火处理后与p SIH-H1质粒连接,转化大肠杆菌DH5α后挑取单克隆并测序;将该克隆进行病毒包装并感染乳腺癌MCF7细胞,用嘌呤霉素进行筛选,最终得到稳定克隆;收取细胞,用Western印迹和q RT-PCR检测ERα在m RNA及蛋白水平上的变化,流式细胞术检测敲低ERα能否影响MCF7乳腺癌干细胞的含量。结果:经测序分析表明目标sh RNA序列成功插入载体,q RT-PCR检测稳定克隆,发现该sh RNA能有效抑制目的基因的m RNA转录水平,同时Western印迹检测发现内源性ERα量明显下降;流式细胞术检测发现ERα敲低的MCF7稳定克隆中雌激素诱导的乳腺癌干细胞含量上升的现象被明显抑制。结论:设计并构建的抑制ERα的sh RNA能够有效抑制ERα的表达,并下调MCF7细胞中乳腺癌干细胞的含量,为研究ERα在乳腺癌干细胞中的功能及机制奠定了实验基础。更多还原

【Abstract】 Objective: To construct a sh RNA interference vector of estrogen receptor α(ERα) gene,and to de-tect its effect on breast cancer stem cells(BCSCs).Methods: Based on human ERα gene sequence,the specificsh RNA oligonucleotide sequences were designed by Invitrogen online software.The synthesized sequences were an-nealed to form double-strand oligonucleotides and cloned into interference vector plasmid p SIH-H1.DNA sequenc-ing confirmed that this sh RNA had successfully been cloned into the p SIH-H1 vector.The sh RNA interferencevector was then cotransfected into 293 T cells with the lentivirus packing vectors.MCF7 cells were infected withthe virus and selected by puromycin.MCF7 cells stably expressing ERα sh RNA were detected by real-time quanti-fication PCR and Western blot.Finally,the effect of inhibition of ERα on BCSCs were detected by flow cytome-try.Results: ERα sh RNA was successfully cloned into the p SIH-H1 vector.q RT-PCR and Western blot showedthat the sh RNA could suppress the expression of ERα.Knockdown of ERα could inhibit estrogen induced BCSCs.Conclusion: The constructed ERα sh RNA interference vector can effectively inhibit the expression of ERα and es-trogen induced BCSCs,which provided a basis for studying the function and mechanisms of ERα in BCSCs.更多还原