鼻咽癌耐药细胞株14-3-3σ表达研究临床检验诊断学

发布时间：2019-07-02 15:11

【摘要】：目的：研究14-3-36基因在鼻咽癌细胞株HNE1和耐顺铂的鼻咽癌细胞株HNE1-DDP中的表达及其启动子区域的甲基化情况,初步探讨14-3-3σ基因与耐药的关系。方法：首先采用蛋白印迹法(Western Blotting)检测H(?)NE1-DDP细胞与HNE1细胞14-3-36的表达,并采用荧光定量PCR(Quantitative PCR,qPCR)检测14-3-36基因的mRNA表达水平。然后利用甲基化抑制剂5-杂氮-2’-脱氧胞苷处理HNE1-DDP细胞,观察不同浓度对细胞生长及细胞周期的影响,并采用Western Blotting及qPCR分别检测药物干预前后14-3-36基因表达的改变。用Western Blotting检测p53蛋白在HNE1及HNE1-DDP中的表达,并采用亚硫酸氢钠处理后测序法(Bisulfite genomic sequencing PCR)检测14-3-36启动子区域CpG位点的甲基化情况。采用SPSS13.0统计软件进行数据分析。结果：较HNE1细胞,14-3-3σ蛋白及其mRNA、p53蛋白在HNE1-DDP细胞株中表达均明显下降。经过不同浓度的5-aza-2'-dC(0,1,5,10μmol/L)处理HNE1-DDP细胞后,细胞生长曲线出现明显差异,细胞活性抑制,细胞阻滞在S期和G2-M期。同时,随着药物的处理,HNE1-DDP细胞的14-3-36蛋白和mRNA重新表达并逐渐上升。此外,亲本细胞株HNE1、耐药细胞株HNE1-DDP及处理后的细胞株中,14-3-3σ启动子区域的CpG位点几乎均为非甲基化状态。结论：14-3-36下调与鼻咽癌耐药相关,提示14-3-36可能是鼻咽癌耐药相关的蛋白。5-aza-2′-dC具有抑制鼻咽癌耐药细胞株生长、阻滞耐药细胞株周期和上调14-3-36的作用,且14-3-36可能也参与了药物的抑制作用。但在鼻咽癌耐药细胞株HNE1-DDP中14-3-36表达下调可能不是由于其甲基化所致,而和抑瘤蛋白p53的表达下降有关,但具体机制有待进一步研究。
[Abstract]:Objective: To study the expression of 14-3-36 gene in HNE1-DDP and its promoter region in nasopharyngeal carcinoma cell line HNE1 and cisplatin, and to investigate the relationship between 14-3-3 gene and drug resistance. Methods: The expression of H (?) NE1-DDP cells and HNE1 cells 14-3-36 was detected by Western Blotting, and the mRNA expression of 14-3-36 gene was detected by fluorescence quantitative PCR (qPCR). And then the HNE1-DDP cells were treated with a methylation inhibitor 5-hybrid nitrogen-2 '-donor cell to observe the effects of different concentrations on cell growth and cell cycle. The expression of 14-3-36 gene was detected by Western Blotting and qPCR, and the expression of p53 protein in HNE1 and HNE1-DDP was detected by Western Blotting. The methylation of the CpG sites in the promoter region of the 14-3-36 promoter was detected by the method of sodium bisulfite treatment. Analysis. Results: The higher HNE1 cells,14-3-3 mitogen and m After treatment of HNE1-DDP cells with different concentrations of 5-aza-2 '-dC (0,1,5,10. mu.mol/ L), the growth curves of the cells were significantly different, the cell activity was inhibited, and the cells were blocked in the S phase and the G2-M phase. The 14-3-36 protein and the mRNA of the HNE1-DDP cell were reexpressed and gradually increased. In addition, the CpG sites of the 14-3-3 gene promoter region of the parent cell strain HNE1, the resistant cell strain HNE1-DDP and the treated cell line were almost non-methyl. Conclusion:14-3-36 down-regulation and nasopharynx The expression of 5-aza-2-dC has the potential to inhibit the growth of the drug-resistant cell line of the nasopharyngeal carcinoma, block the cycle of the drug-resistant cell strain and the up-regulation of 14-3- The expression of 14-3-36 in the HNE1-DDP resistant cell line HNE1-DDP may not be due to its methylation, but the expression of the tumor-inhibiting protein p53 is decreased, but the specific mechanism is to be determined.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R739.63

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