【作者】 刘香丽；

【导师】 张徽；

【作者基本信息】 重庆医科大学 ， 病理学与病理生理学， 2008， 硕士

【摘要】 目的:探讨在乳腺癌细胞中,去甲基化药物5-氮杂脱氧胞苷对雌激素受体α(estrogen receptor alpha, ERα)基因,上皮性钙粘连素(E-cadherin, E-cad)基因表达的影响,及其对细胞增殖和侵袭能力的影响,为乳腺癌的基因治疗提供一定的指导意义。方法:(1)使用去甲基化药物5-氮杂脱氧胞苷处理MDA-MB-231细胞,利用细胞生长曲线、MTT法及流式细胞仪检测5-氮杂脱氧胞苷对MDA-MB-231细胞增殖和周期的影响;(2)通过RT-PCR和免疫组化检测5-氮杂脱氧胞苷对ERα阴性乳腺癌细胞MDA-MB-231 ERα、E-cad及基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达的影响;(3)通过Boyden小室侵袭实验检测5-氮杂脱氧胞苷对乳腺癌细胞浸润能力的影响;(4)用限制性酶PCR检测5-氮杂脱氧胞苷作用前后的乳腺癌细胞中ERα基因启动子及第一外显子区域的甲基化情况。结果:(1)5-氮杂脱氧胞苷能使MDA-MB-231细胞的生长速度明显减慢,细胞周期被阻滞在G0/G1期;(2)5-氮杂脱氧胞苷部分恢复ERα阴性乳腺癌MDA-MB-231细胞ERα及E-cad基因的表达;(3)5-氮杂脱氧胞苷使MDA-MB-231细胞中MMP-2基因表达下降,及该细胞的侵袭能力降低;(4)MDA-MB-231细胞ERα基因中检测到HpaⅡ甲基化位点,5-氮杂脱氧胞苷作用后未检测到甲基化位点。结论:(1)5-氮杂脱氧胞苷可有效抑制乳腺癌细胞生长并导致细胞周期的阻滞,以及明显降低乳腺癌细胞的侵袭能力;(2)5-氮杂脱氧胞苷可以部分的去甲基化和恢复ERα的表达;(3)利用5-氮杂脱氧胞苷可部分恢复ERα和E-cad的表达及降低MMP-2的表达,这可能为其引起乳腺癌MDA-MB-231细胞生物学行为改变的机制之一。更多还原

【Abstract】 Objectiv:To explore the effect of 5-aza-2’-deoxycytidine(5-Aza-CdR) on ERαexpression, E-cad expression, proliferation and invasion of human breast cancer cell line MDA-MB-231, and it could provide instructive value for the gene therapy of human breast carcinoma.Method:1. The breast cancer cell line MDA-MB-231 was treated with 5-Aza-CdR. Cell growth was evaluated by cell counting and MTT assay, and cell cycle was analyzed by flow cytometry. 2. Immunohistochemistry and RT-PCR were used to measure the changes of the ERαexpression, E-cad expression and MMP-2 expression in the MDA-MB-231 cells after 5-Aza-CdR treatment. 3. The invasion ability of MDA-MB-231 cells was tested by Boyden chamber assay. 4. PCR using the extracted DNA and methylation–sensitive restriction enzymes, was performed to detect the methylation of a CpG island in the ERαgene.Results: 1. Compared with the control, the cell growth was significantly reduced and the cell cycle was stuck at G0/G1 phase after treatment. 2. The expression of ERαgene and E-cad gene was partly restored in ERαnegative human breast cancer cell line (MDA-MB-231) after treatment. 3. The expression of MMP-2 in the cell line MDA-MB-231 is downregulated and the invasion ability of MDA-MB-231 cell was decreased after treatment. 4. MDA-MB-231 cell was found methylation-positive in the exon 1 of ERαgene. No methylation was found in 5-Aza-CdR treated cells.Conclusion: 1. The 5-Aza-CdR treatment in MDA-MB-231 cells leads to cell proliferation inhibition, cell cycle arrest and cell invasiveness decline. 2. Methylation was partly reversed and the ERαexpression was partly restored in MDA-MB-231 cells by 5-Aza-CdR. 3. Our findings suggest that the partial restoration of ERαexpression and E-cad expression, and the decrease of MMP-2 expression with 5-Aza-CdR may be a mechanism of biological behaviour change in MDA-MB-231 cells.更多还原