基于诱导多能干细胞技术的子痫前期疾病模型及其发病机理研究

Preeclampsia(PE) is a important cause leading to high morbidity and mortality of both mothers and fetus. The pathogenesis of PE is still not well defined, in which the the disorders of trophoblasts function might be a potential root etiological factor. Establishment of human PE disease cell model for the research on differentiation and invasion of trophoblasts during placentagenesis in vitro will benefit in learning the PE pathogenesis in detail. In this study, hiPSCs will be induced from placental trophoblasts in normal pregnancy women and PE patients. Furthermore, hiPSCs will be differentiated into trophoblasts by BM4 pathway to establish a human cell model of PE. And then the differences in trophoblasts development and invasion are compared between groups. Following, PE associated cytokines will be screened by microarray, and cas9 gene knock-out as well as lentivirus technique is applied in normal hiPSCs to investigate the functional-loss and functional-gain of PE candidate genes. The invasion and vascular remodeling of trophoblasts are detected for a intense study of the underlying molecular mechanism. Meanwhile, sFlt-1,a cytokine which is regarded to be related to the PE symptoms, is evaluated for the potential effect on the development and invasion of trophoblasts in the early stage with a PE-specific hiPSC model. In conclusion, this study will be helpful in understanding the mechanism between disorders of trophoblasts function in early stage and the PE pathogenesis, which will provide a sound evidence for clinical prediction and management in the early stage.

子痫前期（PE）是导致母儿患病和死亡的重要原因。早孕滋养细胞功能异常可能是其根本病因，但机制尚未阐明。建立人PE疾病细胞模型，体外模拟人类胎盘发生过程中的滋养细胞分化和侵袭，将有助于探讨PE机制。本研究拟建立正常妊娠和PE病人胎盘滋养细胞来源的人诱导多能干细胞(hiPSC)，并通过BMP4途径，向滋养细胞诱导分化，建立人PE疾病细胞模型，比较其分化能力和侵袭功能的差异，并通过基因芯片分析，筛选PE相关因子，并进一步用cas9基因敲除、慢病毒等技术对hiPSC进行PE候选基因的功能缺失性或功能获得性研究，观察相应滋养细胞的侵袭与血管重塑功能，并探讨其分子机制。同时，本研究还将利用正常及PE疾病特异性hiPSC研究PE临床症状密切相关因子sFlt-1在早期滋养细胞分化和植入过程中的潜在作用。本研究有利于揭示PE早孕期滋养细胞功能异常的机制，为PE临床早期预测及干预的提供依据。

子痫前期(PE)是导致母儿患病和死亡的重要原因。早孕滋养细胞功能异常可能是其根本病因，但机制尚未阐明。建立人 PE 疾病细胞模型，体外模拟人类胎盘发生过程中的滋养细胞分化和侵袭，将有助于探讨 PE 机制。本研究拟建立正常妊娠和 PE 病人胎盘滋 养细胞来源的人诱导多能干细胞(hiPSC)，并通过 BMP4 途径，向滋养细胞诱导分化，建立人 PE 疾病细胞模型，比较其分化能力和侵袭功能的差异，筛选 PE 相关 因子，并进一步用 cas9 基因敲除等技术对 hiPSC 进行 PE 候选基因的功能缺失性或功能获得性研究，观察相应滋养细胞的侵袭与血管重塑功能，并探讨其分子机制。同时，本研究还将研究 PE 临床症状密切相关因子 sFlt-1 在早期滋 养细胞分化和植入过程中的潜在作用。. 本研究分离了早孕绒毛细胞、正常产妇和子痫前期产妇的胎盘羊膜上皮及间充质细胞，通过非整合、非病毒方式将其成功诱导为hiPS 细胞株，并通过体内、体外实验验证了其干性和多能性。然后利用优化的 BMP4+A83-01+PD173074小分子诱导分化方案定向诱导胚胎干细胞和hiPSC 分化为滋养层细胞，结合 qPCR、免疫荧光染色、ELISA、侵袭实验及基因芯片多种手段证实了分化获得细胞的滋养系特征和生物学功能。在此多能干细胞-滋养层定向分化的细胞模型基础上，探讨了多种因子包括 TEAD4、GATA3和 FOXD3对滋养层早期分化形成的作用，同时还发现ZEB1影响多能干细胞向合体滋养层分化。另外，本项目已建立了 sFlt1敲除的多能干细胞株并且发现 FOXD3跟 PE 具有相关性。. 本项目研究利用胎盘及绒毛等胚外来源细胞成功建立了hiPS 细胞株，而且优化了 BAP 方案建立了多能干细胞-滋养层早期分化的细胞模型。基于该模型已初步揭示了部分重要因子在滋养层早期分化形成中的重要作用，下一步将结合外周血PE 差异化指标在本研究模型中进行深入验证探讨其机制，以及揭示并发现更多的能预测子痫前期的有效易行的标记物。

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