E2F2相关细胞周期调节因子在角膜内皮祖细胞重编程中的作用及其机制研究

Human corneal endothelial cells (HCECs) play a pivotal role in maintaining corneal transparency. Persistent HCEC dysfunction leads to sight-threatening bullous keratopathy. HCECs are notorious for the limited proliferative capacity in vivo and even in vitro. Our previous data showed that activation of LIF-JAK1-STAT3 by MESCM (serum-free), delays contact inhibition of HCECs by upregulation of E2F2 colocalized with nuclear phosphorylated Rb (p-Rb, Ser807/811) with concomitant downregulation of p16INK4a. Moreover, we have recently reported that reprogramming of HCECs to neural crest (NC)-like progenitors can be induced with additional p120/Kaiso knockdown and can obviously promote proliferation of HCEC. Since the said reprogramming can only been found in MESCM, we envision that E2F2 realated cell cycle genes are prerequisite for HCEC reprogramming to NC progenitors. However, the further mechanism is still remains unclear. In this study, we intend to further dissect the mechanism of E2F2 realated cell cycle genes in NC progenitors reprogramming through gene chips and confirm it by in vitro and in vivo activation or inhibition experiments. The ultimate goal of this study is to provide a solid theoretical basis for tissue-engineering corneal construction and corneal endothelium decompensation disease treatment.

人角膜内皮细胞（HCECs）是维持角膜正常透明性的关键，其缺乏将导致致盲性眼病。HCEC在体内缺乏增殖能力，体外其增殖能力也十分有限。我们前期研究表明，不含血清的MESCM培养基可以激活LIF-JAK1-STAT3信号通路引起E2F2表达上调并伴有Rb磷酸化的核转位，同时下调p16的表达从而延迟HCEC接触抑制的发生。并且，通过P120基因敲减处理，我们可以成功的将HCEC重编程诱导为更年轻的神经嵴祖细胞，从而明显提高其体外增殖能力。由于重编程只在MESCM环境中发生，所以我们推测E2F2相关细胞周期调节因子在其中起着关键的调控作用，但其作用机制尚不清楚。本项目拟利用基因芯片技术对E2F2相关细胞周期调节因子进行筛选，并通过上调、阻断试验阐明其在人角膜内皮祖细胞重编程中的具体作用机制，为组织工程角膜内皮植片的构建以及临床角膜内皮细胞失代偿疾病的治疗提供坚实的理论依据。

人角膜内皮细胞（HCECs）是维持角膜正常透明性的关键，其缺乏将导致致盲性眼病。HCEC在体内缺乏增殖能力，体外其增殖能力也十分有限。我们前期研究表明，通过对角膜内皮细胞连接P120基因敲减处理，可以成功的将HCEC重编程诱导为更年轻的神经嵴祖细胞，从而明显提高其体外增殖能力。本课题通过对E2F2相关细胞周期调节因子的研究发现，相比每周的连续基因敲减，单次的P120基因敲减并不能有效诱导重编程，而重编程的诱导中P16INK4a起着关键作用，通过激活和抑制实验证实JAK2-STAT3信号通路作为RhoA-ROCK信号通路的下游信号，在Bmi1介导的角膜内皮祖细胞重编程中发挥重要作用。这些机制的阐明将为组织工程角膜内皮植片的构建以及角膜内皮细胞失代偿疾病的治疗提供新的思路。