5-Aza对肝纤维化进程及miR-29b基因异常甲基化的影响  

Effects of 5-aza-2'-deoxycytidine on the development of liver fibrosis and the abnormal methylation of miR-29b gene

作　　者：林德照 郑建建[2] 林镯[2] 董培红[2] 俞富军[2] 

机构地区：[1]温州市中医院普外科,浙江温州325000 [2]温州医科大学附属第一医院,浙江温州325000

出　　处：《中国卫生检验杂志》2014年第9期1229-1231,1235,共4页Chinese Journal of Health Laboratory Technology

基　　金：国家自然科学基金(81000176/H0317;81100292/H0317);浙江省自然科学基金(Y2090326;Y2110634);王宝恩肝纤维化研究基金(20100002;20120127);温州市科技局资助项目(Y20110033;Y20120127)

摘　　要：目的观察5-氮杂-2'-脱氧胞苷(5-Aza)对TGF-β1诱导的HSC-T6细胞株活化状态及miR-29b基因甲基化的影响。方法采用MTT、qRT-PCR及Western blot技术检测5-Aza治疗组细胞活化状态及miR-29b含量;BSP及TA克隆检测miR-29b启动子区CpG岛甲基化的改变。结果相较TGF-β1组,5-Aza组细胞增殖率下调为58.62%(P<0.05)且Ⅰ型胶原及α-SMA的mRNA及蛋白均显著下降(P<0.05)。miR-29b在5-Aza治疗后呈时间依赖性上升(P<0.05)。BSP及TA克隆显示5-Aza组miR-29b甲基化率为7.27%,呈现下调(P<0.05)。结论5-Aza可负性调控肝纤维化进程,这与miR-29b启动子区去甲基化进程密切相关。Objective To observe the effects of 5- aza- 2＇- deoxycytidine（5- Aza） on the activation state of TGF- β1- induced HSC- T6 cells and the methylation of miR- 29b gene. Methods The effects of 5- Aza on the activation status of TGF- β1- induced HSC- T6 cells were measured by MTT,qRT- PCR and Western blot,and the expressions of miR- 29b were detected in 5- Aza- treated cells at different time. The methylation status of miR- 29b promoter CpG islands in HSC- T6 cells was measured by bisulfite genomic sequencing（BSP） and TA clone. Results The cell proliferation rate in 5- Aza- treated cells was reduced when compared with TGF- β1- treated cells（P〈0. 05）. The mRNA and protein expressions of type Ⅰ collagen and α- SMA also decreased in the 5- Aza group when compared with the TGF- β1group（P〈0. 05）. 5- Aza showed an increase effect on miR- 29b expression in a time- dependent manner after exposure to 5- Aza at 0,24,48 and 72 hours（P〈0. 05）. The average methylation rate of miR-29b promoter CpG islands was 7. 27% in the 5- Aza group,lower than that in the TGF- β1group（P〈0. 05）. Conclusion The effects of 5- Aza showed an negative regulation of liver fibrosis,which were associated with the methylation rate of miR- 29b promoter CpG islands.

关 键 词：肝纤维化 miR-29b 重亚硫酸盐测序PCR TA克隆 

分 类 号：R575.29[医药卫生—消化系统]