补肾安胎法干预miRNAs介导SA蜕膜组织PSG家族基因表达的机制研究

Spontaneous abortion（SA）is the natural termination of pregnancy which occurs at first trimester.Unblanced function of the Decidual stromal cells (DSC) plays an important role during SA .We’ve already comfirm that Minus ShouTai pills which are used to tonifying kidney and preventing miscarriage can protect the function of the DSC. High-throughput sequencing of mRNAs (mRNA-Seq) was used in preliminary study to detect the mRNAs of PSG (Pregnancy specific beta-1-glycoprotein) family which was over-expressed in decidua from women suffering from SA. Microarray analysis was used to detect the miRNAs under-expressed in decidua with the same origin as former. .Therefore , we put forward the hypothesis that the main mechanism of Minus ShouTai pills which can protect the function of decidual cells in SA may mediated by miRNAs that regulate the expression of PSG family .Therefore , based on the theory that kindey is primarily responsible for reproduction, we intend to use DSC cultured in vitro as the target of our systematic study. Largely detect mRNA of PSG gene family and miRNAs and link them with the clinical index .Down regulate and up regulate the miRNAs in DSC with siRNA transfection of DSC to silence the PSG gene family .Then, detect the expression of PSG gene family and the phenotype of DSC. Intervene the DSC .with the extract of Minus ShouTai pills,thus we can inllustrate the mechanism of therapies used for tonifying kidney and preventing miscarriage that was mediated by miRNAs which regulate the expression of PSG family.

自然流产（SA）指妊娠自然终止，多发生在妊娠早期。蜕膜细胞（DSC）功能失衡是SA发生的重要环节。前期研究证实补肾安胎复方对蜕膜细胞功能的保护作用，采用转录组高通量测序（mRNA-Seq）筛选SA蜕膜组织中过表达的PSG基因家族，基因芯片技术筛选同源低表达的miRNAs，经生物信息学分析miRNAs介导PSG基因家族mRNA表达，提出：减味寿胎丸可能通过miRNAs介导SA蜕膜细胞的PSG基因家族mRNA表达而维持妊娠。故根据肾主生殖，以离体蜕膜细胞为载体系统研究：大样本验证SA蜕膜组织中PSG和miRNAs的表达，并与SA临床指标关联；下调正常早孕蜕膜细胞miRNAs，上调SA蜕膜细胞miRNAs并用siRNA沉默PSG基因家族，检测PSG基因家族mRNA和蛋白表达及蜕膜细胞表型变化；减味寿胎丸全提取物干预SA蜕膜细胞，阐释补肾安胎法通过介导miRNAs调控PSG基因家族表达的作用机制。

自然流产（SA）是常见的生殖障碍疾病。前期证实了补肾安胎法对蜕膜细胞和滋养细胞的保护作用，可以提高孕激素受体的表达，减少蜕膜组织的过度凋亡。但具体的凋亡发生机制尚不明确。结合前期研究和生物信息学分析，miR-4653-3p和miR-4684-3p可能介导PSG基因家族基因的表达，提出科学假说：减味寿胎丸可能通过miRNAs介导SA滋养细胞PSG基因家族mRNAs表达而维持妊娠。本研究综合运用分子生物学、病理学、中药药理学等技术从临床样本和细胞层面两方面展开研究。①临床样本：收集50对临床绒毛组织，对PSG1、miR-4684-3p、miR-4653-3p等分子进行验证，并对相关指标进行关联分析。②细胞模型：转染构建细胞模型和RU-486诱导滋养细胞损伤模型，首次明确miR-4684-3p能下调PSG1的表达，增加滋养细胞凋亡从而导致SA的发生。同时运用减味寿胎丸（JW）及其有效成分川续断皂苷Ⅵ（ASD）处理细胞来验证其可通过干预miR-4684-3p上调靶基因PSG1抑制细胞过度凋亡防治SA。已经发表核心文章3篇，培养博士研究生3名。项目负责人入选国家中医药领军人才支持计划，当选为首届岐黄学者。成立广东省教科文卫系统劳模和工匠人才创新工作室:罗颂平劳模创新工作室。创立微信公众号“罗颂平教授工作室”。本研究找到了新的基因miR-4684-3p和PSG1，为临床诊断提供了潜在的标志物。补充了自然流产的发生机制：miR-4684-3p过表达可引起靶基因PSG1下调，介导细胞凋亡的发生同时破坏线粒体膜电位，引起自然流产。阐明了减味寿胎丸及其有效安胎成分川续断皂苷Ⅵ通过抑制凋亡、保护线粒体功能起到安胎作用。丰富了肾主生殖治疗自然流产的作用机制，有利于临床补肾安胎中药的推广。