【作者】 姜黎明；

【导师】 马继延；

【作者基本信息】 华东师范大学 ， 生物医学， 2012， 硕士

【摘要】 目的：探讨细胞朊蛋白(PrPc)在不同因素导致的乳腺癌细胞凋亡中的功能。方法：RNAi逆转录病毒载体进行PrPc表达下调稳定细胞系的构建；免疫荧光的方法对目的蛋白进行定位和半定量；MTT法测定细胞的活性；台盼蓝染色法测定死活细胞的比率；利用Western-bloting方法进行蛋白表达的检测。结果：MDA-MB-435细胞系是雌激素受体阴性且PrPc表达量较高的细胞系,利用RNAi逆转录病毒载体构建了PrPc表达下调的稳定细胞系MDA-MB-435-siPrP(简写成：siPrP)及其对照细胞系MDA-MB-435-siLuc(简写成：siLuc)。 Western-bloting及免疫英光的结果显示经RNA干扰后MDA-MB-435-siPrP细胞系的PrPc的表达被显著的抑制。给予不同的条件处理稳定细胞系,我们发现：各种不同因素导致的细胞凋亡中,PrPc所起的作用不同。(1)在阿霉素处理和葡萄糖剥夺情况下,siPrP细胞系比si Luc细胞系更具有耐受性,说明此种情况下PrPc具有促凋亡功能；(2)在鱼藤酮处理情况下,siPrP细胞系比siLuc细胞系更易于死亡,说明此种情况下PrPc具有抗死亡功能；(3)然而在过氧化氢(H2O2)、紫杉醇及星形孢菌素处理的情况下,siPrP细胞系与siLuc细胞系没有出现明显的差异。为了去除稳定细胞系建系过程中由于抗生素筛选而对细胞系引起的附加效应,我们在瞬时转染siPrP及其对照siLuc的质粒于MDA-MB-435细胞系；转染48小时后,检测转染效率,并重复阿霉素实验,结果发现PrPc下调的细胞系仍有对阿霉素耐受性的现象；表明阿霉素对siLuc和siPrP两种细胞系的不同影响并不是由于稳定细胞系构建过程本身引起的,而是由于PrPc表达下调引起的。随后我们对siPrP细胞系耐受阿霉素处理的机制进行研究,发现阿霉素处理后siPrP细胞系p-p53的表达高于siLuc细胞系；由于MDA-MB-435细胞系的p53是突变体,因此我们在稳定细胞系的基础上转入有转录功能的p53,实验结果显示上调p53后siPrP-p53仍表现对阿霉素的更加耐受性,所以证明了：PrPc在阿霉素处理情况下的抗凋亡功能与p53通路无关。更多还原

【Abstract】 Objective:To investigate the role of cellular prion protein (PrPc) in breast cancer cells apoptosis.Methods:Using Western-bloting method for protein detection; immune fluorescence method for target protein localization and semi quantitative analysis; retroviral vector RNAi for establishing PrPc knockdown stable cell lines; MTT method for the determination of cell viability and normal cell growth curve; trypan blue staining method for determining live/dead cell ratio.Results:The MDA-MB-435cell line is a estrogen receptor negative and PrPc high expression human breast cancer cell line. PrPc knockdown stable cell lines (abbreviated as MDA-MB-435-siPrP:siPrP) and control cell lines (abbreviated as MDA-MB-435-siLuc:siLuc) were successfully constructed using retroviral vectors expressing RNAi targeting PrP or luciferase.Our results revealed that PrPc plays dramatically different roles in cell death caused by various factors.(1) In Doxorubicin treatment or glucose deprivation, siPrP cell line exhibited significantly more tolerance than siLuc cell lines, indicating a pro-apoptotic function of PrPc;(2) however, in rotenone-induced cell death, a higher cell death was observed in siPrP cell line compared to the control siLuc cells, suggesting an anti-apoptotic function of PrPc;(3) in hydrogen peroxide (H2O2), paclitaxel and staurosporine treatments, siPrP cells and siLuc cell lines did not show clear differences, suggesting that PrPc does not play a significant role in these cell death processes. Our results showed that, in the same cell line, PrPc may play different roles in cell deaths induced by various factors. In order to remove the additional effect caused by the stable cell line establishment process due to antibiotic screening of cell lines, we in transiently transfected siPrP and controlled siLuc plasmid to MDA-MB-435cell lines;48hours after transfection, repeat the doxorubicin experiment,results showed that PrPc downregulation of cell lines are still on adriamycin tolerance phenomenon; that of doxorubicin on siLuc and siPrP two cell lines of different effect was not due to stable cell lines construction process, but because of PrPc expression downregulated.Moreover,research on the mechanism of siPrP cell tolerance by doxorubicin treatment, we found that siPrP cell line p-p53expression was higher in siLuc cell line after doxorubicin treatment,; due to MDA-MB-435cell line p53mutants, we transfect widetype p53in stable cell lines, the experimental results show that after up-regulation of p53,siPrP-p53were still more tolerance on doxorubicin treatment,we also showed that the pro-apoptotic function of PrPc in Doxorubicin-treated siPrP cells is p53pathway independent.更多还原