【作者】 黄秋梅；

【导师】 袁洪；

【作者基本信息】 中南大学 ， 药理学， 2009， 硕士

【摘要】 目的:研究谷胱甘肽S-转移酶μ3(glutathione S-transferase mu 3,GSTM3)在乳腺癌中的作用,建立GSTM3稳定过表达或沉默的乳腺癌细胞株MDA-MB-231,初步观察GSTM3对乳腺癌细胞株MDA-MB-231生物学特性的影响。方法:构建GSTM3真核表达重组质粒pcDNA3.1/GSTM3,并利用真核表达载体pSilencer 2.1-U6 neo构建GSTM3特异性siRNA表达质粒;以脂质体转染法将构建好的正确重组质粒和对照载体质粒分别转染MDA-MB-231细胞,RT-PCR和荧光定量PCR法检测GSTM3 mRNA水平,western blot筛选GSTM3蛋白表达改变的稳定细胞株;应用MTT法和细胞划痕运动实验,观察GSTM3对MDA-MB-231细胞体外增殖能力、迁移运动及蒽环类抗肿瘤药物阿霉素敏感性等影响。结果:1.转染GSTM3真核表达质粒,经RT-PCR、荧光实时定量PCR和western实验证明,GSTM3过表达的稳定乳腺癌细胞株MDA-MB-231成功建立。2.转染GSTM3特异性siRNA表达质粒,并经RT-PCR和荧光实时定量PCR实验证明,GSTM3表达沉默的稳定乳腺癌细胞株MDA-MB-231成功建立。3.GSTM3对MDA-MB-231细胞生物学特性的影响:①GSTM3过表达和沉默后对MDA-MB-231细胞体外增殖能力均无明显影响(P＞0.05);②划痕运动试验中,GSTM3表达沉默的MDA-MB-231细胞划痕愈合快(P＜0.05),GSTM3过表达的MDA-MB-231细胞则愈合慢(P＜0.05);③GSTM3过表达后,MDA-MB-231细胞对蒽环类抗肿瘤药物阿霉素的敏感性增加(P＜0.05),GSTM3表达沉默后,MDA-MB-231细胞对阿霉素敏感性降低(P＜0.05)。结论:1.成功构建GSTM3过表达和沉默的乳腺癌细胞株MDA-MB-231。2.GSTM3表达改变对乳腺癌细胞MDA-MB-231体外增殖能力无明显影响,主要改变细胞对阿霉素的药物敏感性,并影响细胞的迁移运动能力,从而改变乳腺癌细胞转移的倾向。更多还原

【Abstract】 Objective:For further study on the role of GSTM3 in breast cancer, MDA-MB-231 breast cancer cell lines in which GSTM3 is overexpressed or silenced were constructed. The effects of overexpressing or silencing of GSTM3 on MDA-MB-231 cells were investigated.Methods:PCR was done to amplify the cDNA sequences of GSTM3 from prepared cDNA. The product was subcloned into pcDNA3.1 mammalian expression vector to obtain recombinant pcDNA3.1/GSTM3. The oligonucleotide sequences encoding the GSTM3 -specific siRNA were designed and subcloned into the vector pSilencer2.1-U6 neo, designated pSilencer-272, pSilencer-363 and pSilencer-529. These recombinant plasmids were transfected into MDA-MB-231 cells respectively. Expression level of GSTM3 mRNA and protein were analyzed by RT-PCR, real-time quantitative PCR and western blot. Using cell proliferation, cell migration assay, then the effect of GSTM3 on MDA-MB-231 cells proliferation and motility were investigated in vitro. Growth inhibition of ADM on cells was measured by the MTT assay.Results:1. Analyzed by semi-quantitative RT-PCR, real-time RT-PCR and Western blot, overexpression of GSTM3 was detected in cells transfected with pcDNA3.1/GSTM3.2. Analyzed by semi-quantitative RT-PCR and real-time RT-PCR, GSTM3 expression was significantly decreased in the siRNA interfered cell lines.3. The effects of GSTM3 overexpression or silenced on the breast cancer cell line MDA-MB-231:①GSTM3 overexpression or silencing did not alter proliferation of MDA-MB-231 cells in vitro.②MDA-MB-231 cells motility was inhibited significantly by GSTM3 overexpression, and accelerated by GSTM3 silencing.③Overexpression of GSTM3 increased inhibitory effect of ADM on MDA-MB-231 cells, but the effect was reversed by GSTM3 silencing. Conclusion:1. MDA-MB-231 cell lines in which GSTM3 are stably overexpressed or silenced has been obtained.2. GSTM3 overexpression inhibited MDA-MB-231 cells motility, and enhanced inhibitory effect of ADM on MDA-MB-231 cells. And GSTM3 silencing promoted cells motility, and decreased inhibitory effect of ADM on MDA-MB-231 cells. The results indicated that GSTM3 might be associated with invasiveness and chemosensitivity of breast cancer cells.更多还原