TSP1诱导角膜创伤愈合瘢痕形成的机制研究

Corneal scarring due to cornea surgery, trauma, and infection is the main reason for corneal blindness. Our previous studies have found that thrombospondin1 (TSP-1) which was not exist in normal cornea stroma being highly expressed during corneal wound healing, and may have a key role in corneal scar formation. To analyze the mechanism of TSP1 on corneal scar formation, a natural rat cornea wound healing model and a three-dimensional culture cornea wound healing model using acellular porcine cornea matrix (APCM) will be produced by laser photorefractive keratectomy (PRK) respectively to detect the expression of TSP1 and its correlations with corneal scarring formation. And then, the TSP1/TGF-β1 activity and expression, related signaling pathways, keratocytes morphology transformation and function will be discussed by using corresponding antagonists intervention study in a three-dimensional culture model of rat keratocytes with APCM. Next, a rat corneal wound intervention healing model will be made by PRK, to verify the molecular mechanisms of TSP1-TGF-β1 signaling pathways on corneal scarring formation. This study will provide a new theoretical basis and therapeutic target for corneal scarring during cornea wound healing.

角膜盲仅次于白内障居世界第二位，而手术、创伤及感染等所致的角膜瘢痕形成，是角膜盲的主要原因。我们前期研究发现正常角膜基质内不表达的血小板反应蛋白1（TSP-1）高度表达于角膜创伤修复的整个过程，且与 角膜瘢痕形成的关键因子TGF-β1在空间和时间上的表达呈平行关系，提示TSP1参与诱导角膜瘢痕形成，且可能通过TGF-β1通路发挥重要作用。本课题首先通过准分子激光角膜切削术（PRK）制作大鼠角膜及脱细胞猪角膜基质（APCM）三维培养角膜创伤自然愈合模型，明确TSP1的表达来源及其对角膜基质重塑瘢痕形成的作用；再利用APCM三维细胞培养，探讨TSP1是否通过TGF-β1通路对角膜瘢痕形成及基质细胞功能转化产生效应及其作用机制；最后制作大鼠角膜创伤愈合干预模型，验证TSP1-TGF-β1通路对于角膜创伤瘢痕形成的作用机制。本研究将有望给角膜创伤愈合瘢痕化研究提供新的理论依据和治疗靶点。

角膜盲发病率仅次于白内障居世界第二位，而手术、创伤及感染等所致的角膜瘢痕形成，是角膜盲的主要原因，而角膜移植是目前治疗角膜盲的唯一有效手段。因此阐明角膜瘢痕形成的机制，体外构建具有正常角膜结构和功能的角膜替代物，是防治角膜盲的关键。本课题通过优化体外三维培养条件，应用脱细胞猪角膜基质（APCM）三维构建了具有天然角膜结构和特征的角膜板层替代物，并成功用于角膜板层移植，随访观察半年，该构建物能够和受体角膜有效融合，证实该构建物能够同于角膜修复。通过准分子激光角膜切削术（PRK）制作角膜创伤自然愈合模型，研究发现在角膜创伤修复过程中，TSP1与TGF-β1表达时间空间曲线与角膜基质细胞形态功能改变及瘢痕形成呈正相关关系，瘢痕愈合过程中角膜基质细胞活化向肌成纤维细胞转化，TGF-β1通路相关瘢痕因子cTGF、Tenasin-C等表达量增加，基质细胞标志物ALDH3A1表达量减少。本研究证实TSP1及TGF-β1通路在角膜创伤愈合修复中的作用以及体外构建的角膜替代物具有一定的角膜创伤修复能力，为角膜创伤修复的防治提供了实验基础。