【作者】 单云峰；

【导师】 谢维； 樊红；

【作者基本信息】 东南大学 ， 遗传学， 2006， 硕士

【摘要】 目的:通过检测DNMT3B (DNA methyltransferase 3B,DNMT3B)在不同肝癌细胞系中的表达水平和分析DNMT3B表达抑制所诱导的下游基因的表达及癌细胞增殖凋亡情况,探讨DNMT3B和肝癌发生的关系。方法:应用实时荧光定量RT-PCR (Real-Time Fluorescent Quantitative RT-PCR)方法分析正常肝细胞系、肝癌癌旁细胞系及7个肝癌癌细胞系中DNA甲基转移酶表达水平,同时分析肝癌细胞系SMMC-7721中DNMT3B表达抑制所诱导的发育和肿瘤发生相关的基因SNCG,PDCD4和MBD3的表达,并以MSP(Methylated Specific PCR,MSP)方法检测SNCG基因的启动子区甲基化状态。以流式细胞仪技术检测DNMT3B表达抑制对细胞增殖力及凋亡率的影响。结果:DNMT1,DNMT3A,DNMT3B在所检测的肝癌细胞系中均存在高表达的趋势,尤其DNMT3B在肝癌细胞系中的表达水平明显高于肝癌癌旁和正常肝细胞系,提示DNA甲基转移酶的高表达可能与肝癌发生有关。DNMT3B表达抑制诱导了与发育和肿瘤发生相关基因SNCG、MBD3、PDCD4的表达,对其中的SNCG基因的启动子区甲基化状态检测未发现明显的改变,表明DNMT3B表达抑制并未引起SMMC-7721细胞中SNCG基因的启动子区去甲基化,其高表达可能存在其他的机制。DNMT3B表达抑制,肿瘤细胞的增殖力明显受到抑制,G0-G1期延长,凋亡率增高。结论:DNMT3B在肝癌细胞中的高表达可能与肝癌的发生有关。DNMT3B表达抑制诱导了某些肿瘤相关基因的表达,这种诱导并不完全依赖于DNMT3B对CpG位点的甲基化作用。抑制DNMT3B表达可降低肝癌细胞系SMMC-7721的增殖能力。更多还原

【Abstract】 Objective: To explore the relationship between DNMT3B and hepatocellular carcinogenesis by detecting the expression of DNMT3B in hepatocellular carcinoma cell lines and analyzing tumor-related genes in DNMT3B knocked-down hepatocellular carcinoma cell line.Methods: Real-Time Fluorescent Quantitative RT-PCR was used to detect the expression of DNMTs in normal liver cell line, cell line established from pericarinoma tissue and hepatocellular carcinoma cell lines. Upregulated tumor-related genes induced by DNMT3B RNAi such as SNCG,PDCD4 and MBD3 were also analyzed by Real-Time Fluorescent Quantitative RT-PCR in hepatocellular carcinoma cell lines. The methylation status of SNCG was detected by MSP (Methylated Specific PCR ). The cell cycle and rate of apoptosis were analyzed by Flow Cytometry in DNMT3B knocked-down hepatocellular carcinoma cell lines.Results: In hepatocellular carcinoma cell lines DNMTs is expressed at a high level compared to that in pericacinoma cell line and normal liver cell line, especially DNMT3B. The expression of tumor-related genes such as SNCG, PDCD4 and MBD3 was induced by DNMT3B RNAi in hepatocellular carcinoma cell line and there was no change of methylation status in CpG island of SNCG promoter. The ability of cell proliferation was suppressed and the rate of apoptosis was increased in DNMT3B knocked-down hepatocellular carcinoma cell line.Conclusion: The over-expression of DNMT3B in hepatocellular carcinoma cell lines may be related to hepatocellular carcinogenesis.The repression of DNMT3B induced the expression of some tumor-related genes and influenced the proliferation ability of SMMC-7721 by some ways.更多还原