妊娠期高血压疾病FABP4 DNA 甲基化及特异性miRNAs调控机制的研究

Hypertensive disorder complicating pregnancy(HDCP) is a chronic inflammatory disease whose clinical manifestation is a transient increase of blood pressure in pregnancy. Its main feature is extensive vascular endothelial damages caused by systemic small artery spasm. However, it has not been reported that if HDCP is caused by DNA methylation aberration of FABP4 which is the cross-target gene between inflammation and lipid metabolism. Preliminary studies have found that hypermethylation and hypomethylation co-exist in different genes of HDCP, and miRNAs is an important way of gene expression regulation, which suggest the existence of a deeper level regulation mechanism. Thus this project intends to copy the HDCP model, uses real-time PCR and so on to detect the changes of FABP4 in placenta, and verifies its effects in HDCP by its silence and over-expression in cells. The changes of FABP4 DNA methylation and regulatory factors such as DNMTs in placenta are detected by high-throughput MethyLight assay in order to clarify the DNA methylation mechanism in HDCP, and to determine the key targets by using block strategies such as RNA interference. To screen HDCP-specific miRNAs by using microarray analysis. Transfecting miRNAs inhibitor expression vector and the recombinant plasmidand carrying DNMTs. This project seeks to explore the targeted regulation mechanism of DNMTs through specific miRNAs and to identify the key targets so as to provide a theoretical basis for the targeted therapy of HDCP.

妊娠期高血压疾病(HDCP)是以全身小动脉痉挛致血管内皮广泛损伤为主要特征的一过性血压升高的慢性炎症性疾病，是否通过炎症和脂代谢交叉靶基因FABP4 DNA甲基化异常引起HDCP未见报道,前期研究发现HDCP中不同基因DNA高低甲基化并存, 且miRNAs是基因表达调控重要方式，提示存在更深层次调控机制,因此本项目拟复制HDCP动物模型，实时PCR等分析胎盘中FABP4的变化，在细胞中使其沉默和过表达，验证其在HDCP中作用；高通量MethyLight法等检测胎盘组织中FABP4 DNA甲基化和DNMTs等调控因子变化，阐明HDCP中DNA甲基化的作用机制，采用RNA干扰等阻断策略确定关键靶点；运用微阵列技术筛选HDCP特异性miRNAs，转染miRNAs抑制物表达载体和携载DNMTs重组质粒，探讨特异性miRNAs靶向调控DNMTs的机制，确定关键靶点，为HDCP靶向治疗提供理论依据。

背景：妊娠期高血压疾病(HDCP)是孕妇因妊娠后内环境改变而导致的以一过性血压升高、全身小动脉痉挛等致血管内皮广泛损伤为主要特征的严重危害母婴健康的疾病， 是引起孕产妇和围生儿死亡的主要原因之一。 .内容：复制HDCP动物模型，实时PCR等分析胎盘中FABP4的变化，细胞中使其沉默和过表达，验证其在HDCP中的作用；检测胎盘中FABP4 DNA甲基化和DNMT1等调控因子变化，阐明HDCP中DNA甲基化的作用机制，确定关键靶点，筛选作用于DNMT1的特异性miRNAs,分别转染miRNAs抑制物表达载体和DNMTs重组质粒，探讨特异性miRNAs靶向调控DNMTs机制。.结果：妊娠第19天，与对照组比较，妊高症组大鼠血压和尿蛋白均升高，TC和FFA显著升高(P<0.05)；Western Blot检测FABP4、DNMT1的表达，显示FABP4表达升高，DNMT1降低(P<0.05)；100μmol·L-1 L-NAME干预后FABP4启动子荧光素酶活性增加最明显，加入AZC后，其活性增加更明显(P<0.05)；ntMS-PCR检测FABP4启动子区DNA甲基化改变，结果100μmol·L-1 L-NAME作用的FABP4发生低甲基化改变最明显，且过表达DNMT1后显示FABP4甲基化程度升高(P<0.01))；通过生物信息学分析提示调控DNMT1的特异性miRNA为miR-148a/152。分别将野生型和突变型DNMT1 3’UTR质粒与miR-148a/152的minic共转染，显示DNMT1萤光素酶活性分别为下降和无改变。.该项目共发表SCI 7篇，专著2本。.意义：该课题通过锚定调控HDCP的关键基因，以DNA甲基化为突破点探讨其调控机制，为防治HDCP提供新靶点、带来新突破。其实施将有利于阐明HDCP的分子机制，寻找致病分子和致病环节，确定关键靶点，为HDCP的靶向治疗提供新的干预途径。