HhIP基因调控EMT在DBP致尿道下裂发生中的作用及其甲基化对疾病预测的研究

Abstract：As the starting of two-child policy, demands on promoting good prenatal and postnatal care is getting more pressing. Di-n-butyl phthalate (DBP) is a well-known environmental endocrine disruptors (EEDs) which has drawn wide attention because of toxicity in reproductive development. And the lack of effective methods to eliminating deformities during pregnancy after exposuring to EEDs remains a tough challenge. In our previous research, we investigated the differential expression of gene in genital tubercles(GT) of DBP induced hypospadiac male rat and undeformed male rat. We found the significant increase HhIP in hypospadiac GT as well as the weaken epithelial mesenchymal transformation (EMT) and decreased levels of methylation promoter of HhIP. It is reported that HhIP could inhibit HH signaling pathway and futher attenuate EMT which has been found a important role in urethral fusion. Therefore, we hypothesized that the DBP induced abnormal expression of HhIP promotes the deveopment of hypospadias through the process of EMT. The methylation status of HhIP promoter region indicates the activity of the gene and the occurrence of the abnormality. This project intends, in cell and animal levels, to illuminate the role EMT which is regulated by HhIP in DBP innduced hypospadias and investigate the practicability of prognosing deformity by testing methylated level of HhIP in amniotic fluid through gene transfection and interference，molecular biological detection combined morphological observation.

二胎政策的启动对优生优育提出了更高要求。环境内分泌干扰物邻苯二甲酸二丁酯（DBP）生殖发育毒性引起了广泛关注。环境暴露后孕期缺乏有效的排畸手段。前期实验中，我们利用表达谱芯片检测DBP孕期染毒后所致尿道下裂、染毒未畸形子鼠生殖结节（GT）中基因的表达差异，结果发现尿道下裂子鼠GT中HhIP基因表达明显上升，EMT减弱，同时伴随着HhIP基因启动子甲基化水平的下降。已有研究表明HhIP可能通过抑制HH信号通路减弱细胞EMT，后者在尿道融合中起着重要的作用。我们认为DBP染毒后HhIP对尿道上皮细胞EMT调控引起了尿道下裂的发生，HhIP启动子区域甲基化状态标记着基因的活性以及畸形的是否发生。故本研究在前期研究基础上通过基因转染和干预、分子生物学检测结合形态学观察在动物和细胞层面阐明HhIP对EMT的调控在DBP致尿道下裂发生过程中的作用以及对羊水中HhIP的甲基化检测预测畸形发生的可行性。

本研究通过检测孕期DBP暴露的仔鼠生殖结节中Snail、E-cadherin、N-cadherin、β-Catenin等EMT相关基因和蛋白的表达水平，进一步明确了DBP染毒致尿道下裂的作用机制。同时，通过对仔鼠生殖结节进行IHC分析，检测LC3 II、LC3 I、mTOR、p-mTOR、Beclin1等自噬相关基因及Gpx1、Cat、GR、Gst等抗氧化基因的表达水平，以及DBP处理后尿路上皮细胞内Ca2+浓度等，我们发现：EMT异常致使尿道下裂受氧化应激作用及自噬的异常激活调控，而体内及体外实验对Hedgehog信号通路相关基因的检测结果表明，Hedgehog的抑制会与自噬的异常激活密切相关，通过Affymetrix 3’IVT 表达谱芯片检测尿道下裂和正常对照雄性子鼠生殖结节中基因谱的表达差异结果提示，IP3R表达上调导致尿路上皮细胞氧化应激。此外，通过对全身多脏器大体、病理HE染色等形态学分析发现：DBP暴露导致纤维化肾脏中RhoA/ROCK通路异常激活、TGF-β表达上调和EMT的异常激活，而在DBP处理大鼠肾小管上皮细胞（NRK-52E）后，ROS的产生使TGF-β1表达上调，进而促进Snail调控的EMT激活，而ROCK抑制剂Y-27632治疗仔鼠8周后，仔鼠肾脏EMT明显受到抑制，肾纤维化程度显著降低，肾功能显著改善。我们还发现，孕期DBP暴露可能导致仔鼠肾小管上皮细胞中HhIP来调节Hedgehog信号通路从而诱导自噬，该机制可能在肾纤维化发展中同样起重要作用。此外，我们还发现孕期DBP染毒不仅影响子代多脏器发育，还会加重孕鼠内分泌疾病，如孕期糖尿病（GDM）。课题组成功构建妊娠糖尿病大鼠模型，检测胰腺中FoxM1和pSTAT1的表达水平，禁食试验和OGTT测定血糖及胰岛素分泌水平得知：DBP可通过其毒理学效应影响GDM的进展，显著增加pSTAT1的表达并抑制FoxM1，导致β细胞活力下降。