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目的:探讨5种食管鳞癌细胞株中p16基因转录失活以及去甲基化药物对其作用的机制。方法:采用细胞培养、PCR、DHPLC、MSP、Northern印迹、MTT等方法,检测EC1,EC18,EC109,TE1,TE10食管鳞癌细胞株中p16基因的缺失、突变、甲基化状态和p16的转录表达,观察去甲基化药物5脱-氧氮杂胞苷对p16基因转录表达和细胞增殖能力的影响。结果:除了EC1,其余4种食管鳞癌细胞株均检测出p16基因的不同变化:纯合缺失(EC18),纯合型甲基化(EC18,EC109),杂合型甲基化(TE1,TE10),且纯合缺失的发生率较低(20%),甲基化的发生率较高(80%),经过5-Aza-CdR处理,可见2株发生了杂合型甲基化的TE1和TE10细胞p16基因的转录表达得到了逆转。结论:不同食管鳞癌细胞株p16基因转录失活的原因是不同的,启动子区甲基化为主要原因,其中杂合型甲基化可以被去甲基化药物5-脱氧氮杂胞苷逆转,使p16的转录表达上调。
Objective: To investigate the transcriptional inactivation of p16 gene in 5 kinds of esophageal squamous cell carcinoma and the mechanism of its demethylation. Methods: The deletion, mutation, methylation status and p16 transcription of p16 gene in EC1, EC18, EC109, TE1 and TE10 esophageal squamous cell carcinoma cell lines were detected by cell culture, PCR, DHPLC, MSP, Northern blot and MTT Expression and to observe the effect of demethylation drug 5-desoxacin on the transcription and expression of p16 gene and cell proliferation. Results: In addition to EC1, the other four esophageal squamous cell carcinoma cell lines were detected in different changes of p16 gene: homozygous deletion (EC18), homozygous methylation (EC18, EC109), heterozygous methylation (TE1, TE10), and the incidence of homozygous deletion was lower (20%) and the methylation rate was higher (80%). After 5-Aza-CdR treatment, 2 strains had heterozygous methylation Transcriptional expression of p16 gene in TE1 and TE10 cells was reversed. CONCLUSION: The transcriptional inactivation of p16 gene in different esophageal squamous cell carcinoma cell lines is different, the promoter methylation is the main reason, in which heterozygous methylation can be demethylated 5-deoxy-azacytidine Reverse, so that p16 transcriptional expression increased.