PIB5PA在增强三阴性乳腺癌细胞MDA-MB-231对紫杉醇的敏感性中的作用及其机制研究

发布时间：2018-07-02 10:43

本文选题：乳腺癌 + PIB5PA　；参考：《安徽医科大学》2015年硕士论文

【摘要】：研究背景和目的：乳腺癌是女性常见的恶性肿瘤,其中有15%称为三阴性乳腺癌(Triple-negative breast cancer,TNBC),即不表达雌激素受体、孕激素受体和人表皮生长因子受体2。目前国内外对TNBC的研究进展较为缓慢,化疗仍然是其患者术后治疗的主要手段。紫杉醇作为抗微管化疗药物,是TNBC术后的首选临床化疗药物,然而临床应用中仍有很大部分TNBC对此耐药,其机制可能与TNBC细胞内PI3K/Akt信号通路激活有关。PIB5PA可抑制PI3K/Akt信号通路的活化,但其是否能通过此途径来增强三阴性乳腺癌细胞对紫杉醇的敏感性尚无明确的答案,也是该课题的研究重点。方法：体外培养乳腺癌细胞株MCF-7和MDA-MB-231,采用MTT比色法观察不同浓度(0,0.1,0.2,0.3,0.4,0.5μmol·L-1)紫杉醇处理后对两种细胞株生存率的影响；流式细胞仪PI单染法检测不同浓度紫杉醇处理MCF-7和MDA-MB-23148h后对细胞凋亡率的影响；应用免疫蛋白印迹法(Western blot)检测I0.3μmol·L-1紫杉醇处理MCF-7和MDA-MB-231不同时间点Bcl-2家族成员蛋白表达情况。将pCGN-PIB5PA质粒转染对紫杉醇相对不敏感人乳腺癌细胞MDA-MB-231,同时设立对照组,0.3μpmol·L-1紫杉醇处理24h后,免疫蛋白印迹法检测PIB5PA,磷酸化Akt以及Bcl-2家族成员蛋白的表达情况,采用MTT法和PI单染法检测PIB5PA转染MDA-MB-231后对紫杉醇敏感性的影响。结果：选取两株乳腺癌细胞株MCF-7和MDA-MB-231经过紫杉醇一起培养后,发现紫杉醇能明显诱导MCF-7细胞的凋亡,然而MDA-MB-231细胞株对紫杉醇相对不敏感；与此现象相对应的是,紫杉醇诱导MCF-7细胞中Bim蛋白的明显上调,而MDA-MB-231细胞株经紫杉醇处理后Bim表达几乎没有变化；过表达PIB5PA后三阴性细胞株MDA-MB-231对紫杉醇敏感性明显增强,表现为转染后MDA-MB-231细胞的生存率明显降低,同时其凋亡率明显升高；同时伴随着细胞中磷酸化Akt水平降低,Bim的表达增高。结论：PIB5PA能显著增强紫杉醇诱导MDA-MB-231细胞的凋亡；PIB5PA在MDA-MB-231细胞凋亡中的作用可能与抑制磷酸化脂,上调Bim的表达有关。
[Abstract]:Background and objective: breast cancer is a common malignant tumor in women, 15% of which are called triple-negative breast cancer TNBC, that is, estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. At present, the progress of TNBC research at home and abroad is slow, chemotherapy is still the main method of postoperative treatment. Paclitaxel, as an anti-microtubule chemotherapeutic drug, is the first choice of clinical chemotherapeutic drugs after TNBC surgery. The mechanism may be related to the activation of PI3K / Akt signaling pathway in TNBC cells. PIB5PA can inhibit the activation of PI3K / Akt signaling pathway, but there is no clear question whether PI3K / Akt signaling pathway can enhance the sensitivity of triple-negative breast cancer cells to paclitaxel. It is also the research focus of this subject. Methods: breast cancer cell lines MCF-7 and MDA-MB-231 were cultured in vitro. The survival rate of breast cancer cell line MCF-7 and MDA-MB-231treated with paclitaxel was observed by MTT colorimetry. The apoptotic rate of MCF-7 and MDA-MB-23148 h treated with different concentrations of paclitaxel was detected by flow cytometry Pi single staining, and the expression of Bcl-2 family members was detected by Western blotting assay (blot) at different time points treated with I0.3 渭 mol L-1 paclitaxel and MDA-MB-231 at different time points. PCGN-PIB5PA plasmid was transfected into human breast cancer cell line MDA-MB-231, which was relatively insensitive to paclitaxel, and the expression of PIB5PA, phosphorylated Akt and Bcl-2 family members were detected by Western blot after treated with paclitaxel at 0.3 渭 pmol L-1 for 24 hours. MTT assay and Pi single staining were used to detect the effect of PIB5PA transfection on the sensitivity of paclitaxel to MDA-MB-231. Results: two breast cancer cell lines MCF-7 and MDA-MB-231 were cultured with paclitaxel. It was found that paclitaxel could induce apoptosis of MCF-7 cells, but MDA-MB-231 cell line was relatively insensitive to paclitaxel. Bim protein was up-regulated in MCF-7 cells induced by paclitaxel, but the expression of Bim in MDA-MB-231 cells was almost unchanged after treatment with paclitaxel, and the sensitivity of MDA-MB-231 cells to paclitaxel was significantly increased after overexpression of PIB5PA. The results showed that the survival rate and apoptosis rate of MDA-MB-231 cells decreased significantly after transfection, and the level of phosphorylated Akt decreased with the increase of Bim expression in MDA-MB-231 cells. Conclusion the effect of PIB5PA on apoptosis of MDA-MB-231 cells induced by paclitaxel may be related to the inhibition of phosphorylated lipid and the up-regulation of Bim expression in MDA-MB-231 cells.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R737.9

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