非酶糖基化交联水凝胶基底对角膜成纤维细胞行为影响的体外研究

发布时间：2018-03-04 20:27

本文选题：非酶糖基化交联　切入点：胶原水凝胶　出处：《太原理工大学》2017年硕士论文　论文类型：学位论文

【摘要】：圆锥角膜是临床常见的眼科疾病。针对圆锥角膜的治疗方法有多种,其中角膜胶原交联术(corneal collagen cross-linking,CXL)能够有效阻止圆锥角膜进展,倍受青睐。CXL术后角膜基质的胶原纤维结构发生变化,应力状态重新分布,基质细胞生存的力学微环境发生了改变,细胞如何适应这种变化、并与之相互作用,是我们亟待解答的问题。非酶糖基化交联,在实现基质交联环境的同时可与细胞共凝,因此为体外研究CXL术后角膜基质细胞与基质相互作用提供了良好的载体。本文通过添加核糖对胶原水凝胶实现非酶糖基化交联,探讨了胶原交联后圆锥角膜成纤维细胞形态、铺展、增殖以及细胞骨架的重排,为研究CXL术后角膜微环境改变对角膜基质细胞生物学行为的影响提供参考。论文主要工作如下:1.采用不同核糖浓度(0、50、100、150、200 mM)对鼠尾I型胶原进行非酶糖基化交联,通过自行搭建的胶原薄膜力学特性检测系统检测了胶原水凝胶薄膜的弹性模量,采用共聚焦显微镜和扫描电镜观测了胶原微观结构。研究表明:随着核糖浓度的增加,胶原力学性能得到了一定改善,从180 Pa提高到470 Pa;胶原纤维的直径变粗。胶原水凝胶薄膜的力学性能和胶原纤维直径变化之间呈正相关性。2.原代提取培养圆锥角膜成纤维细胞,将其接种于胶原水凝胶的表面,采用FDA-PI双色荧光法观测细胞活性;通过浮雕显微镜观察细胞形态变化;通过Image plus5.1软件对细胞铺展面积进行测量;采用WST-1检测对细胞增殖情况;通过对细胞核与细胞骨架的共染观察细胞骨架的重排情况,并测量了细胞核面积的变化。研究表明:随着核糖浓度的增加,细胞形态发生明显改变,细胞铺展面积以及细胞核面积变小;细胞活性未受到明显的影响,细胞的增殖随着核糖浓度的增加而降低;生长于低核糖浓度交联的水凝胶基底上的细胞肌动蛋白应力纤维更加明显。我们的研究表明非酶糖基化交联后的胶原水凝胶力学微环境发生明显变化,这种改变对圆锥角膜成纤维细胞的铺展、增殖及骨架重排等细胞行为均产生了一定的影响,适度的交联在提高胶原水凝胶力学性能的同时亦适合细胞的生长。这将为探索CXL术后角膜基质细胞与基质相互作用提供有价值的参考。
[Abstract]:Keratoconus is a common clinical ophthalmic disease. There are many treatments for keratoconus, among which corneal collagen cross-linking can effectively prevent keratoconus from progressing. The structure of collagen fibers in corneal stroma was changed, the stress state was redistributed, and the mechanical microenvironment of stromal cells was changed after CXL. How could the cells adapt to this change and interact with it? Nonenzymatic glycosylation and cross-linking can be achieved in matrix crosslinking environment at the same time with cell coagulation. Therefore, it provides a good carrier for studying the interaction between stromal cells and stromal cells after CXL in vitro. In this paper, the morphology of keratoconus fibroblasts after collagen crosslinking was studied by adding ribose to the collagen hydrogel for non-enzymatic glycation crosslinking. Spreading, proliferation, and cytoskeleton rearrangement, In order to study the effect of corneal microenvironment changes on the biological behavior of corneal stromal cells after CXL, the main work of this paper is as follows: 1. The elastic modulus of collagen hydrogel film was measured by a self-built mechanical property test system. The microstructure of collagen was observed by confocal microscope and scanning electron microscope. The results showed that the elastic modulus of collagen hydrogel film increased with the increase of ribose concentration. The mechanical properties of collagen were improved from 180 Pa to 470 Pa, the diameter of collagen fibers became thicker, the relationship between the mechanical properties of collagen hydrogel membrane and the diameter of collagen fibers was positive. 2. The primary extraction and culture of keratoconus fibroblasts, The cells were inoculated on the surface of collagen hydrogel, and the cell activity was observed by FDA-PI two-color fluorescence method. The morphological changes of cells were observed by relief microscope, the cell spreading area was measured by Image plus5.1 software, the proliferation of cells was detected by WST-1. The rearrangement of cytoskeleton was observed by co-staining of nucleus and cytoskeleton, and the change of nuclear area was measured. Cell spreading area and nuclear area became smaller, cell activity was not significantly affected, cell proliferation decreased with the increase of ribose concentration. The cellular actin stress fibers grown on the hydrogel substrate with low ribonose concentration were more obvious. Our results showed that the mechanical microenvironment of collagen hydrogels after non-enzymatic glycation crosslinking was significantly changed. These changes have a certain effect on the proliferation, proliferation and cytoskeleton rearrangement of keratoconus fibroblasts. Proper crosslinking not only improves the mechanical properties of collagen hydrogel, but also suits the growth of cells, which will provide a valuable reference for exploring the interaction between corneal stromal cells and stroma after CXL.
【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.2

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