眼部细胞穿膜肽重组神经生长因子跨角膜上皮细胞转运机制的研究

Many corneal surgery and corneal diseases can cause corneal nerve damage. Drug-induced corneal nerve regeneration is practical clinical needs and considered as the key for successful corneal nerve repairment. The vast majority of the proteins which promote the axon growth including nerve growth factor(NGF) belong to macromolecules. They can not directly transport into the corneal stroma by corneal epithelial cells which plays a role in the obstruction of corneal nerve repairment. Peptide for ocular delivery (POD) is one of cell-penetrating peptide (CPPs). As a polypeptide carrier, POD can transport macromolecules across epithelium cells. This project intends to recombine exogenous NGF and POD to form a recombinant nerve growth factor (POD-NGF) and to detect the transport level of inter-cellular of corneal epithelial cells. Then, fluorescent labeling and endocytosis inhibitor method will be used to compare the impact on transport of POD-NGF with or without endocytosis inhibitors. Therefore, to clearly transport mechanism across corneal epithelial cells of POD-NGF. Subsequently, the role of POD-NGF on corneal nerve repairment will be evaluate in animal models of corneal nerve damage . so as to promote the development of corneal nerve growth drugs, Overcall, findings of this research may porvide new ideas to find a new way to promote corneal nerve regeneration.

许多角膜手术和角膜疾病会造成角膜神经损伤，用药物来帮助神经修复是临床现实需求。绝大多数促进神经轴突生长的蛋白包括神经生长因子（NGF）都属于大分子物质，不能直接通过角膜上皮细胞进入角膜基质层发挥促进角膜神经修复的作用。眼部细胞穿膜肽（peptide for ocular delivery, POD）是细胞穿膜肽 (cell-penetrating peptides, CPPs)的一种，作为一种多肽载体，可以实现跨细胞转运大分子物质。该课题拟将POD和外源性NGF重组形成眼部细胞穿膜肽重组神经生长因子（POD-NGF），检测其穿透角膜上皮细胞实现跨细胞转运的水平。通过荧光标记并使用胞吞作用抑制剂的方法，对比胞吞作用抑制剂使用与否对POD-NGF入胞作用的影响，明确POD-NGF的入胞机制，并在角膜神经损伤的动物模型上评价POD--NGF对角膜神经损伤修复的作用。从而为开发药物提供基础依据

许多角膜手术和角膜疾病会造成角膜神经损伤，用药物来帮助角膜神经修复是临床现实需求。绝大多数促进神经轴突生长的蛋白包括神经生长因子（NGF）都属于大分子物质，不能直接通过角膜上皮细胞进入角膜基质层发挥促进角膜神经修复的作用。眼部细胞穿膜肽（peptide for ocular delivery, POD）是细胞穿膜肽 (cell-penetrating peptides, CPPs)的一种，作为一种多肽载体，可以实现跨细胞转运大分子物质。本研究成功的进行了质粒的构建，质粒构建后，通过将质粒转染表达菌株进行前期蛋白的制备。由于蛋白质和核酸的性质差异很大，我们在试验中发现，直接制备POD-NGF融合表达直白蛋白不可行，后尝试做重组表达。经反复试验，调整各项实验参数，得到纯化蛋白眼部穿膜肽重组神经生长因子POD-NGF。纯化蛋白的产量稀少，课题组开始尝试进行124ml Erlen Flask方案进行质粒转染，调整技术路线以得到更多产量的纯化蛋白。同时进行的是动物模型的建立和验证。课题组以化学损伤和经上皮准分子切削角膜的方式实现动物角膜神经损伤模型。达到了为进一步制备促进角膜神经修复的药物提供基础研究的目的。