完全性葡萄胎血管发生障碍中外泌小体的影响及机制

Complete hydatidiform mole (CHM) is one of the main gestational trophoblastic diseases. CHM is the product of an abnormal pregnancy with swollen chorionic villi and hyperplasic trophoblasts, but without mature vessels. However, immuno-fluorescence with CD34 and other vessels markers prove, there does exist vasculogenesis during the early villi development stage, but unable to mature for defects in further development. The villious vessels are generally believed differentiated from mesenchymal stem cells (MSCs), while the differentiation processes of MSCs are regulated by numerous molecules with complex signal pathway at different levels. Moreover, the differentiation of MSCs is dependent on the microenvironment they located. Exosomes are small (40–100 nm) membrane vesicles of endocytic origin that are released into the extracellular environment and are involved in cell-to-cell communication. Exosomes are important extracellular transporters of miRNAs, and they sorting miRNA automatically by hnRNPs and then functionally transferred to recipient cells. Our experiments with exsomes isolated from villous stroma cultures or CHM hydatidiform liquid proved the exosomes from CHM hydatidiform liquid inhibit the angiogenic sprouting and capillary lumen formation progress by human umbilical vein endothelial cells (HUVEC).In this project, we plan to isolate exosomes from CHM hydatidiform liquid, CHM patient serum and villous stroma cultures. Using PKH67 labeling, we would observe the dynamic process of exosomes absorption by MSCs and HUVEC. After miRNA high-throughout sequencing and protein mass spectrometry sequencing, we would isolate the angiogenesis and vasculogenesis associated differential expressed proteins and miRNAs. Next, we will construct the lenti-viral vectors of those proteins and infect MSCs or HUVEC to study the effects on the differentiation to vessels of MSCs and effects on the capillary lumen formation of HUVEC. Besides, we will package the differential miRNA mimics to exosomes and further to study their effects on MSCs or HUVEC. Finally, we will investigate the pathways or targets of those miRNAs involved in. Our findings will be important for revealing the mechanism of vessels development defects in CHM and rich scientific theory for other trophoblastic diseases with villious abnormality, such as FGR and PIH.

完全性葡萄胎(CHM)绒毛发育早期存在少量的血管生成，但因发育障碍未能成熟。外泌小体是胞外重要的miRNA载体，能够主动分选包装miRNA。绒毛血管来源于间充质干细胞（MSCs），而MSCs的分化严格依赖于微环境。我们的前期研究表明，与正常绒毛间质细胞培养上清外泌小体相比CHM囊液中的外泌小体能够显著抑制原代脐静脉内皮细胞（HUVEC）的血管形成能力。故本项目拟在分离CHM囊液、CHM患者血清以及正常绒毛间充质培养上清中的外泌小体基础上，分别进行miRNA测序和质谱实验，筛选出差异性的血管生成相关蛋白和miRNA。进而分别在MSCs和HUVEC上研究差异蛋白和miRNA对其向血管分化、发育的影响，并进一步对差异miRNA参与的信号通路及靶基因进行预测和验证。本项目不仅可以深入揭示CHM血管发生障碍的机理，也为其他存在绒毛血管发育异常的疾病如FGR、PIH等发病机制提供有力的理论指导。

完全性葡萄胎存在滋养细胞的异常增殖和血管发育障碍，而外泌小体是胞外重要的RNA传递载体。本项目研究取得以下发现：第一，葡萄胎囊液外泌小体能够抑制HUVEC的血管形成以及诱导绒毛间充质干细胞的凋亡。第二，与滋养细胞肿瘤相比，miR-371a-5p、miR-518a-3p和miR-21在完全性葡萄胎中低表达，而miR-371a-5p、miR-518a-3p和miR-21能够促进滋养细胞的增殖和运动。我们首次证明了BCCIP和MST1分别为miR-371a-5p和miR-518a-3p的靶基因。KEGG富集分析表明，miR-371a-5p调控VEGF、TGF-β和gap junction通路，miR-518a-3p调控p53、细胞周期和细胞凋亡等信号通路。同时，我们还确认了PDCD4和PTEN为miR-21的靶基因，并发现miR-21主要调控Akt 473位丝氨酸的磷酸化水平。此外，我们还发现miR-371a-5p和miR-518a-3p在完全性葡萄胎来源的外泌体中低表达，而miR-21高表达。第三，PDCD4在正常早孕绒毛、HM、侵袭性葡萄胎组织组织中表达水平依次降低，同时PDCD4能够调节滋养细胞的增殖以及通过调控MMP3、MMP8、ITGB2、TIMP1、TIMP2调节滋养细胞的运动。第四，与正常绒毛间充质来源外泌体相比，完全性葡萄胎来源的外泌小体中LncRNA ANKRD10-IT1和Linc01278显著下调，而Linc01420显著上调，其具体作用机制仍在进一步研究中。本项目结果为阐明绒毛相关疾病的发生和发展具有重要的意义，鉴定的miRNA对于滋养细胞疾病的预后也具有一定的临床价值。