具有生物活性白血病抑制因子（LIF）全蛋白质水凝胶人角膜内皮细胞膜片的构建及功能研究

Severe shortage of available corneal donors remains a global challenge. Tissue- engineered cornea is a potential method to solve this problem, in which tissue-engineered corneal endothelium is the key. The proliferative ability of human corneal endothelial cell is strictly limited in vitro and in vivo, which may be due to cell contact inhibition making cell stay in G1 phase. Previous studies suggested that leukemia inhibitory factor (LIF) can delay corneal endothelial cell contact inhabitation by LIF-JAK1-STAT3 pathway and increase the ability of cell proliferation. Recombinant protein based-hydrogels have favorable biocompatibility and biodegradability. Various bioactive growth factors like LIF can be introduced by genetically encoding the genes and expression, which allows versatile interaction with cells. It can be used to precisely simulate the characteristics of biological tissue and cell microenvironment for the control of cell behavior and fate. This study will apply the protein hydrogel to human corneal endothelial cell regeneration. We plan to introduce bioactive LIF into protein hydrogels, construct engineered grafts, explore the role in delaying cell contact inhabitation and the proliferation of human corneal endothelial cells, and prove its curative effect in bullous keratopathy. This study provides a platform and can also extended to other bioactive factors as well. The success of this study may provide a new approach to solve the lack of donor corneas.

角膜供体材料的匮乏仍然是一个全球性的问题。组织工程角膜是解决该问题的潜在途径。而组织工程内皮材料的研发成为彻底解决该问题的关键。人角膜内皮细胞体内外增殖能力有限，可能与细胞接触抑制使细胞处于G1期有关。既往研究认为白血病抑制因子（LIF）可通过LIF-JAK1-STAT3通路延迟角膜内皮细胞接触抑制，增加细胞增殖能力。基于重组蛋白质的水凝胶具有良好的生物相容性和降解性，允许通过编码引入丰富多样的活性因子与细胞相互作用，可以精确模拟生物组织的特性与细胞微环境，进而控制细胞的行为和命运。本课题拟将蛋白质水凝胶应用于人角膜内皮细胞再生，计划通过基因编码和表达在蛋白质水凝胶中引入生物活性LIF因子，构建植片，在此基础上研究其延迟细胞接触抑制、并促进人角膜内皮细胞增殖的能力，及对大泡性角膜病变的治疗效果。该研究亦可引入其他活性因子，是一个普适的技术手段，有望为解决角膜供体材料匮乏提供一个新的途径。

角膜供体材料的匮乏仍然是一个全球性的问题。组织工程角膜是解决该问题的潜在途径。.人角膜内皮细胞体内外增殖能力有限，可能与细胞接触抑制使细胞处于G1期有关。既往研究认为白血病抑制因子（LIF）可通过LIF-JAK1-STAT3通路延迟角膜内皮细胞接触抑制，增加细胞增殖能力。本项目检测了LIF、CS5-LIF融合蛋白对人原代角膜内皮细胞的作用；构建了以包被LIF基质为载体的角膜内皮细胞植片，并检测对大泡性角膜病变的治疗效果；采用含LIF培养基扩增的人原代角膜内皮细胞进行细胞治疗对大泡性角膜病变的治疗效果；并比较两种方案的效果。研究发现LIF可以促进人原代角膜内皮细胞增殖；CS5-LIF融合蛋白可抑制人角膜内皮细胞的纤维化；包被LIF基质为载体的角膜内皮细胞植片对治疗大泡性角膜病变的有效且安全；含LIF培养基扩增的人原代角膜内皮细胞对治疗对大泡性角膜病变的有效且安全；比较两种方案的治疗效果，发现细胞治疗起效快、炎症反应轻。研究中申请2项专利，均已经获得实用新型专利授权。根据本项目研究结果可知LIF有助于角膜内皮细胞体外扩增、以基质为载体的角膜内皮细胞植片安全有效、细胞治疗可获得更好效果并可摆脱支架材料的束缚，临床治疗前景更明朗。