表没食子儿茶素没食子酸诱导人恶性淋巴瘤CA46细胞系p16基因去甲基化及转录  

Demethylation and Transcription of p16 Gene in Malignant Lymphoma Cell Line CA46 Induced By EGCG

作　　者：喻爱芳[1] 沈建箴[1] 陈志哲[1] 范丽萍[1] 林福安[2] 

机构地区：[1]福建医科大学附属协和医院血液科、福建省血液病研究所,福建福州350001 [2]福建医科大学附属漳州市医院血液科,福建漳州363000

出　　处：《中国实验血液学杂志》2008年第5期1073-1078,共6页Journal of Experimental Hematology

基　　金：福建省自然科学基金资助项目(编号C0540014);福建医科大学教授基金资助项目(编号JS06080)

摘　　要：本研究旨在探讨表没食子儿茶素没食子酯酸(epigallocatechin-3-gallate,EGCG)诱导人恶性淋巴瘤CA46细胞系p16基因去甲基化及转录激活的可能机制。以生长曲线、MTT法检测EGCG对CA46细胞生长和增殖的抑制作用;利用流式细胞仪DNA含量分析法探讨EGCG对CA46细胞周期的影响;采用巢式甲基特异性PCR法(nested-methylation specific PCR,n-MSP)及DNA克隆测序分析法检测EGCG作用前后CA46细胞p16基因甲基化状态;应用RT-PCR检测p16、DNA甲基转移酶(DNMT3A、DNMT3B)基因mRNA的表达。结果表明:与对照组相比,3组不同浓度EGCG均能明显抑制CA46细胞生长,G0/G1期细胞增加;不同浓度EGCG作用48小时后p16基因甲基化程度减弱;未处理组细胞p16基因呈微弱表达,未用药组、不同浓度EGCG(6、12、24)μg/ml处理组条带灰度值与β-actin1比值分别为(0.05±0.01)、(0.19±0.03)、(0.39±0.10)、(0.85±0.09),各处理组p16基因mRNA表达明显高于未用药组,其差异有显著意义(p<0.05);与对照组相比,EGCG作用48小时后CA46细胞甲基转移酶DNMT3A、DNMT3B mRNA的表达均下降。结论:EGCG可能通过抑制甲基转移酶DNMT3A、DNMT3B和(或)直接对p16基因去甲基化,使p16基因mRNA表达上调,恢复其活性,从而实现其对细胞周期的调控功能,将细胞阻滞于G0/G1期,抑制CA46细胞的增殖。The study was purposed to investigate the possible mechanism of epigallocatechin-3-gallate（EGCG） induced p16 gene demethylation and transcription regulation in the malignant lymphoma cell line -CA46. The induced growth inhibition of CA46 cells was assayed by growth curve and MTT; the DNA content of CA46 cells was analyzed by flow cytometry after being exposed to EGCG; the methylation status of the p16 gene in CA46 cell line before and after treatment with EGCG was detected by the nested-methylation specific PCR and DNA sequencing; the mRNA of p16 and DNA methyltransferases （ DNMT3A and DNMT3B ） gene were determined by RT-PCR. The results showed that in comparison with the control, all the 3 different concentration of EGCG were able to inhibit the growth of malignancy cell lines and increase the cell number in G0/G1 phase. After treatment with EGCG for 48 hourse, the methylation level was apparently attenuated in a concentrationdependent manner. Expression of p16 gene in untreated group was mild while in the treated groups it had been greatly strengthened,as compared with untreated group, the gray scale ratio of p16 to β-actin 1 treated with EGCG （6, 12, 24） μg/ ml was increased from （ 0. 05 ± 0. 01 ） to （0. 19 ± 0. 03 ）, （0. 39 ±0. 10 ）, （0. 85 ± 0. 09） respectively, exhibiting a significant difference （p 〈 0. 05 ） ; as compared with the untreated group, after treatment with EGCG for 48 hourse, the expressions of DNMT3A and DNMT3B were obviously down-regulated. It is concluded that EGCG can activate and upregulate the expression of p16 gene mRNA which inhibits the proliferation of CA46 cell through inducing the G0/G1 arrest by demethylation and/or by inhibiting DNMT3A and DNMT3B gene.

关 键 词：表没食子儿茶素 淋巴瘤 CA46细胞 p16基因去甲基化 

分 类 号：R733.1[医药卫生—肿瘤] R979.1[医药卫生—临床医学]