Wnt信号通路和YAP交互作用促进人胚胎干细胞定向分化为角膜内皮细胞的机制研究

Corneal disease is a major cause of blindness. Due to the shortage of cadaveric corneas, the conventional corneal transplantation is difficult to meet the clinical requirements. But the tissue-engineered corneas are hopeful to replace the cadaveric corneas for corneal transplantation. The source of corneal endothelium is a difficult point in the field of tissue-engineered corneas. The previous studies have shown that human corneal endothelial cells(hCECs) induced by human embryonic stem cells(hESCs) have low differentiation efficiency and poor cell activity. The “cross-talk” between Wnt signaling pathway and transcriptional co-factor YAP may play a key role in solving this problem. We will take APCM as a three-dimensional scaffold material, hESCs as seed cells. Using CRISPR/Cas9 and ChIP technology, we will try to analyze the regulation mechanisms of "cross-talk" between Wnt signaling pathway and YAP on the differentiation from human embryonic stem cells into corneal endothelial cells. We will also investigate the effects of interaction between Wnt and YAP on the biological behavior of grafted hCECs in the rabbit model of corneal endothelial dysfunction. This project will be helpful for elucidating the molecular mechanisms of differentiation from hESCs into hCECs and provide new molecular regulation targets for cell replacement therapy of corneal blindness.

角膜病是严重的致盲性眼病，角膜移植为主要治疗途径但受限于供体紧缺。组织工程角膜有望替代天然角膜成为供体材料。角膜内皮的来源是组织工程角膜研究难点。目前诱导人胚胎干细胞（hESCs）分化为人角膜内皮细胞（hCECs）存在诱导效率低、细胞活性差等问题。Wnt信号通路和转录激活辅助因子YAP的交互作用在hESCs来源的人眼周神经嵴干细胞（pNCSCs）分化为hCECs阶段起关键作用，但作用机制尚不清楚。本项目拟采用改良的APCM材料为三维支架，hESCs为种子细胞，采用CRISPR/Cas9、ChIP等技术，阐明Wnt与YAP相互作用对体外三维培养pNCSCs定向分化为hCECs的调控机理，并在角膜内皮功能不全兔模型检测Wnt与YAP对hESCs来源的hCECs移植后生物学行为的影响。研究结果有助于阐明hESCs分化为hCECs的分子机理，为角膜盲的细胞替代治疗提供新的理论依据。

角膜病是严重的致盲性眼病，角膜移植为主要治疗途径但受限于供体紧缺。组织工程角膜有望替代天然角膜成为供体材料。角膜内皮的来源是组织工程角膜研究难点。目前诱导人胚胎干细胞（hESCs）分化成人角膜内皮细胞（hCECs）存在诱导效率低、细胞活性差等问题。Wnt信号通路和转录激活辅助因子YAP的交互作用在hESCs来源的人眼周神经嵴干细胞（pNCSCs）分化为hCECs阶段起关键作用，但作用机制尚不清楚。本项目体外试验部分，采用hESCs为种子细胞，成功将hESCs诱导为pNCSCs，通过Qpcr研究显示，神经嵴相关基因PAX3,PAX7,SOX9,MSX1以及SNAI2等表达显著升高，干细胞基因OCT4,KLF4以及NANOG等表达显著降低，Wnt通路相关表达基因SP5，AXIN2表达显著升高；在pNCSCs分化诱导为hCECs的过程中，加入抑制Wnt信号通路调控因子DKK2可以促进hCECs的生成。在体试验部分，完成了大鼠角膜内皮中YAP的表达定位，研究显示YAP的表达分布于细胞膜、细胞质以及细胞核；完成了大鼠从出生到成年（PO-P180），角膜内皮中YAP的表达变化，研究发现，YAP随着大鼠年龄的增长其表达逐渐减弱，且主要在细胞核上表达。利用物理损伤建立了不同损伤面积的角膜内皮功能不全大鼠模型，并在角膜内皮功能不全大鼠模型检测YAP对hCECs再生的影响，结果表明YAP在角膜内皮细胞损伤后高表达。研究结果有助于阐明hESCs分化为hCECs的分子机理，为角膜盲的细胞替代治疗提供新的理论依据。