重组人角质细胞生长因子-2克隆表达及其对角膜损伤修复机制研究

发布时间：2018-05-02 02:03

本文选题：角质细胞生长因子-2 + 克隆　；参考：《吉林大学》2010年博士论文

【摘要】： 角质细胞生长因子-2(KGF-2)是成纤维细胞生长因子家族的一员。KGF-2对上皮组织具有高度特异性,可加快上皮及基质细胞的修复,且不导致胶原的过度增生,对瘢痕的减少有积极的意义。虽然角膜细胞可分泌一定量的KGF-2,但及时修复仍需补充外源KGF-2,以促进角膜上皮细胞及基质细胞的增殖和移行。因此,开发KGF-2产品,研究其对角膜损伤的作用及机制,对于拓展KGF-2临床应用具有重大意义。本研究利用大肠杆菌原核表达系统成功构建了KGF-2工程菌株,对KGF-2发酵及纯化工艺进行了研究,利用正交实验设计筛选到KGF-2滴眼液的保护剂配方,建立了KGF-2体外生物学活性的测定方法,以日本白兔碱烧伤模型和激光损伤模型研究了KGF-2体内促角膜损伤修复作用及机制。动物实验结果显示,应用不同浓度KGF-2进行治疗后,1周时各种损伤表现均较损伤对照组明显减轻,角膜层厚度接近正常,水肿基本消退,且无明显的炎症反应;伤后2周无炎症反应复发,角膜上皮细胞生长良好,排列整齐,基质层内有较多胶原纤维,板层纤维排列整齐且与角膜上皮平行排列,与正常角膜结构没有明显差别,实验结果提示KGF-2可明显减轻角膜基质水肿,抑制角膜基质成纤维细胞过度增生,抑制炎性细胞浸润,有利于角膜上皮细胞的再生与修复。阳性对照药bFGF治疗组在治疗2周后角膜上皮过度增生呈波浪状,表面凹凸不平,基质中有较多成纤维细胞增生,且板层纤维亦呈波浪状。本实验结果提示,KGF-2可通过加速角膜上皮细胞的再生和迁移,促进角膜基质纤维修复等机制,促进碱烧伤和激光烧伤后受损角膜的修复,对角膜损失具有很好的治疗作用。本课题研究为KGF-2在眼科临床的应用奠定了基础。
[Abstract]:Keratinocyte growth factor (KGF-2) is a member of fibroblast growth factor family. KGF-2 is highly specific to epithelial tissue, can accelerate the repair of epithelial and stromal cells, and does not cause excessive proliferation of collagen, which has positive significance for the reduction of scar. Although corneal cells can secrete a certain amount of KGF-2, it is necessary to supplement exogenous KGF-2 in order to promote the proliferation and migration of corneal epithelial cells and stromal cells. Therefore, it is of great significance to develop KGF-2 products and to study its effect and mechanism on corneal injury in order to expand the clinical application of KGF-2. In this study, KGF-2 engineering strain was successfully constructed by using E. coli prokaryotic expression system. The fermentation and purification of KGF-2 were studied. The protective agent formula of KGF-2 eye drops was screened by orthogonal design. A method for determining the biological activity of KGF-2 in vitro was established. The effects and mechanisms of KGF-2 on corneal injury in vivo were studied with alkali burn model and laser injury model of Japanese white rabbit. The results of animal experiments showed that all kinds of injuries were significantly alleviated at 1 week after treatment with different concentrations of KGF-2, corneal thickness was close to normal, edema basically disappeared, and there was no obvious inflammatory reaction. There was no recurrence of inflammatory reaction at 2 weeks after injury. Corneal epithelial cells grew well and arranged neatly. There were more collagen fibers in stromal layer and lamellar fibers were arranged neatly and parallel to corneal epithelium. There was no significant difference between corneal epithelial cells and normal corneal structure. The results suggest that KGF-2 can significantly reduce corneal stromal edema, inhibit the excessive proliferation of corneal stromal fibroblasts, inhibit the infiltration of inflammatory cells, and facilitate the regeneration and repair of corneal epithelial cells. After 2 weeks of treatment, the corneal epithelial hypertrophy in the bFGF group was wavy, the surface was uneven, there were more fibroblasts proliferation in the matrix, and the lamellar fibers were also wavy. The results suggest that KGF-2 can accelerate the regeneration and migration of corneal epithelial cells, promote corneal stromal fiber repair, promote the repair of damaged cornea after alkali burn and laser burn, and have a good therapeutic effect on corneal loss. This study lays a foundation for the clinical application of KGF-2 in ophthalmology.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R772.21

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