ES微环境通过调控端粒酶通路增强角膜缘干细胞干性及其抑制凋亡机制的深化研究

The embryonic stem cell micro-environment plays an important role in postponing the somatic cells senescence and promoting their proliferation. Our previous work has demonstrated that the proliferative capacities of rabbit corneal epithelial cells and human corneal endothelial cells were promoted by embryonic stem cell-conditioned medium, based on this, combining that our findings that telomerase rejuvenated in the cytoplasm of corneal epithelial cells cultured in embryonic stem cell-conditioned medium, we propose to confirm and consummate our hypothesis that functional properties of stem-like corneal epithelial cells can be enhanced by co-culturing with embryonic stem cells via activation of the integrinβ1-FAK-PI3K/Akt signaling pathway from the three following aspects:1. to verify whether ES micro-environment maintains or regains stemness of human limbal stem cells and inhibits their apotosis via the maintenance of telomerase activity; 2. to confirm the roles of telomerase and FAK/Wnt signaling pathways in the maintenance of biological characteristics in human limbal stem cells; 3. to figure out the definite effects of telomerase-p21-mitochondrial axis in inhibiting apoptosis in human limbal stem cells. This project will focus on the scientific problem "the stemness maintenance in human limbal stem cells", aim to disclose the essential role of ES-micro-environment based on the mechanism of the regulation of telomerase activities, and to illustrate the mechanism of telomerase signaling pathways enhancing ES micro-environment and promoting and maintaining functional properties of adult stem cells.

ES微环境具有延缓体细胞衰老，促进其增殖的作用。我们在发现ES条件培养液可促进兔角膜上皮与人角膜内皮细胞增殖的基础上，结合在年轻化细胞胞浆中表达端粒酶的现象，拟从三个方面进一步验证和完善我们提出的ES微环境通过激活integrinβ1- FAK- Akt信号通路加强共培养细胞干性的假设：1.确定ES微环境是否通过调控端粒酶活性维持或恢复人角膜缘干细胞（LSCs）干性、抑制其凋亡；2. 验证端粒酶与调控FAK/Wnt信号通路在LSCs生物学特性维持中的作用；3.明确端粒酶-P21-线粒体通路抑制凋亡的作用。本项目紧紧围绕"LSCs干性维持"这一科学问题，从端粒酶调控机制入手，旨在揭示ES微环境作用的本质，阐明端粒酶信号通路增强ES微环境促进和维持成体干细胞特性的机制。

ES微环境具有延缓体细胞衰老，促进其增殖的作用。我们在发现ES条件培养液可促进兔角膜上皮与人角膜内皮细胞增殖的基础上，结合在年轻化细胞胞浆中表达端粒酶的现象，进一步验证了ES微环境通过激活integrinβ1- FAK- Akt信号通路加强共培养细胞干性的假设：以CnT-20培养液为对照组，CnT-20+20%ES(ESC-CM)为实验组，进行人角膜缘干细胞（human limbal stem cells, LSCs）的培养，克隆形成试验检测细胞增殖。对ESC-CM组细胞进行RNA 干扰，阻断端粒酶的表达，流式细胞仪检测干扰前后细胞凋亡、线粒体膜电位(Mitochondrial Potential, Δψm)的情况；免疫荧光、流式细胞仪检测干扰前后端粒酶表达、活性氧(Reactive Oxygen Species,ROS)的改变；利用RT-PCR、免疫荧光、Western blot对P63、ABCG2、integrinβ1、CK3等mRNA及蛋白表达进行比较；Western blot检测FAK、pFAK、Akt、pAkt、GSK3β、pGSK3β、P21蛋白的改变。结果显示与CnT-20组相比，ESC-CM组的LSCs 细胞具有更强的增殖能力、更多的传代数量(可以传到8代)、更高的克隆形成能力与更高的干细胞标记达水平。ESC-CM通过促进端粒酶活性的维持增强了LSCs细胞的未分化状态、抑制其凋亡，这是通过ROS产生的减少、Δψm 的维持与P21表达的降低来实现的。我们还验证了ES微环境体内应用具有安全性和有效性，体外构建了具有活性的组织工程角膜，探究了ES环境降低肿瘤恶性程度的可能机制，进行了角膜缘干细胞微环境重建以及角膜抗原伪装研究，完善了ES微环境的研究。