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目的:探讨膀胱癌细胞中DR4、DR5甲基化状态以及与肿瘤坏死因子相关凋亡诱导配体(TRAIL)耐受间的关系。方法:采用RT-PCR技术检测膀胱癌T24细胞、BIU87细胞表面DR4、DR5mRNA表达,对低表达或无表达DR4、DR5膀胱癌细胞采用甲基化特异性PCR(MSP)检测DR4、DR5启动子甲基化状态。同时应用流式细胞仪检测TRAIL组以及TRAIL+5-氮杂胞苷组细胞凋亡状态。结果:RT-PCR示膀胱癌T24细胞无DR4、DR5表达,MSP法研究显示:DR4、DR5基因呈甲基化状态,对TRAIL诱导的凋亡表现为耐受状态(100ng/mlTRAIL,凋亡率7.6%),经去甲基化试剂5-氮杂胞苷处理,T24细胞DR4、DR5表达随时间而增加,100ng/mlTRAIL,凋亡率达29.2%;BIU-87细胞表达DR4、DR5并对TRAIL敏感(100ng/mlTRAIL凋亡率30.4%),应用5-氮杂胞苷处理BIU87细胞,DR4、DR5表达无差异,100ng/mlTRAIL,细胞凋亡率31.7%,MSP法检测DR4、DR5基因呈非甲基化状态。结论:膀胱癌T24细胞DR4、DR5启动子区域甲基化与TRAIL耐受有关,去甲基化可逆转上述耐受状态,可为TRAIL的下一步临床应用提供基础研究资料。
Objective: To investigate the relationship between the methylation status of DR4 and DR5 in bladder cancer cells and the tolerance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Methods: The expression of DR4 and DR5 mRNA in bladder cancer T24 cells and BIU87 cells was detected by RT-PCR. The expressions of DR4 and DR5 mRNA in DR4 and DR5 bladder cancer cells were detected by methylation-specific PCR (MSP) Base state. At the same time, the apoptosis of TRAIL group and TRAIL + 5-azacytidine group were detected by flow cytometry. Results: RT-PCR showed no expression of DR4 and DR5 in bladder cancer T24 cells. MSP assay showed that DR4 and DR5 genes were methylated and resistant to TRAIL (100ng / ml TRAIL, apoptosis rate 7.6%). After demethylation reagent 5-azacytidine treatment, the expression of DR4 and DR5 in T24 cells increased with time, and the apoptosis rate was 100%. The apoptotic rate was 29.2% in 100ng / mlTRAIL. The expression of DR4 and DR5 in BIU-87 cells was TRAIL was sensitive to TRAIL (30.4% apoptosis rate of 100ng / ml TRAIL), the BIU87 cells were treated with 5-azacytidine, the expression of DR4 and DR5 was no difference, 100ng / ml TRAIL, the apoptosis rate was 31.7% Unmethylated status. CONCLUSION: Methylation of DR4 and DR5 promoter regions in bladder cancer T24 cells is associated with TRAIL tolerance. Demethylation can reverse the above tolerance status and provide basic information for the next clinical application of TRAIL.