TGF-β诱导乳鼠上皮组织DNA甲基转移酶及相关miRNA表达的研究

TGF-β Induced Abnormal Expression Level of DNA Methyltransferase and Related miRNA During Neonatal Rat Colon Tissue Culture

目的建立大鼠(乳鼠)肠上皮组织体外培养实验体系,检测TGF-β干预后大肠组织DNA甲基转移酶(DNA methyltransferase,Dnmt)及相关mi RNA表达的变化,研究TGF-β诱导上皮组织的相关机制。方法取出生3天的SD大鼠大肠组织建立体外组织块培养体系,观察组织块生长的情况,再用10 ng/ml TGF-β干预组织生长,对比干预前后组织生长状况的变化,并在干预不同的时间取组织样本;HE染色观察组织结构变化,提取组织块中RNA,采用RT-PCR方法检测DNA甲基转移酶及其相关mi RNA的表达水平变化。结果 TGF-β处理不同时间的组织块HE染色分析结果表明,在培养过程中,组织基本结构逐渐不能保持。对Dnmts及相关mi RNA的表达水平检测结果表明,TGF-β干预组织后,Dnmts及相关mi RNA的表达水平异常,相应升高或降低;在TGF-β干预过程中,使用甲基转移酶抑制剂5-Aza-d C干预24 h后,Dnmts的表达水平下降至无法检出,而此时相关mi RNA的表达水平相应升高,甚至恢复至接近无TGF-β干预状态。结论组织块培养过程中加入TGF-β干预,组织块的生长情况、Dnmts及相关mi RNA的表达水平表现出异常,甲基化转移酶抑制剂能够抑制Dnmts的表达并且调节mi RNA异常表达。

Objective To build an in vitro neonatal rat epithelial tissue culture system, to detect the expression level of DNA methyltransferase（Dnmt） and relevant mi RNA in the cultured tissues, and to investigate related mechanism of induction effect of TGF-beta during colon tissue culture. Methods We applied 3-day-old SD rat＇s intestinal tissues to build the in vitro tissue culture system and observed the growth state, added 10ng/ml TGF-beta in culture system to induce the growth of cultured tissues. The tissues from different time were collected in the experiment and the morphological change of the tissues was observed by HE staining through microscope. The expression levels of DNA methyltransferase and related mi RNA were detected by reverse transcription（RT）-PCR. Results Slices of the tissues from different time were fi xed, embedded and stained with Hematoxylin and Eosin. The morphology of the cultured tissues was changed as the extension of the culture time and the epithelial structure gradually disintegrated. RT-PCR results showed that after treated with 10ng/ml TGF-beta, the expression levels of Dnmts and relevant mi RNA were abnormal; Dnmts expression were up-regulated or down-regulated; after TGF-β and 5-Aza-d C interference, the express level of Dnmts couldn＇t be detected; meanwhile the relevant mi RNA expression level was up-regulated, even close to untreated status. Conclusion TGF-β could infl uence the growth of colon tissues, lead to the abnormal expression levels of Dnmts and relevant mi RNA; 5-Aza-d C inhibits the expression of Dnmts and regulates the abnormal expression of mi RNA.