力学刺激对兔角膜基质细胞增殖及bFGF表达的影响

发布时间：2018-05-30 08:03

本文选题：LASIK + 周期性拉伸　；参考：《太原理工大学》2012年硕士论文

【摘要】：准分子激光原位角膜磨镶术(laser in situ keratomileusis,LASIK)是目前开展较为广泛且应用于临床效果良好的治疗近视的角膜屈光手术。每年有近百万人接受LASIK手术治疗。LASIK手术主要通过改变角膜中央光学区的曲率,使屈光不正现象得以改善。此手术在角膜瓣下的基质层中进行,这样做既保存了角膜上皮层和前弹力层的完整度,同时又可防止角膜上皮下雾状浑浊(Haze)的发生。 LASIK术后角膜损伤修复过程是多因素参与的复杂过程。其中,力学环境的改变对LASIK术后角膜组织的修复是一个不可忽视的因素。LASIK术后角膜层变薄,在相同的眼内压作用下,角膜所受的应力明显增大,角膜成纤维细胞的力学环境发生了变化,可能影响其生长、增殖。碱性成纤维细胞生长因子(basic Fibroblast Growth Factor,bFGF)既能促进伤口愈合与受损组织的修复还能促使受损细胞发生相应的形态学改变。因此研究在体及离体不同力学环境下,角膜组织及角膜成纤维细胞中bFGF的表达与分布,以及力学刺激对兔角膜基质细胞增殖的影响,探究力学因素在LASIK术后角膜损伤修复中的作用,为临床治疗近视提供有力的理论依据和指导。本文的主要研究内容及结论如下： 1.建立不同切削量的LASIK手术动物模型,使在体角膜处于不同力学环境中,采用免疫荧光染色观察角膜上皮及基质层中bFGF表达；苏木素-伊红(HE)染色观察角膜基质细胞增殖的变化。研究发现：bFGF在上皮及基质层中均有表达,但基质层中荧光强度总体高于上皮层,70%一周组基质层荧光强度明显强于上皮层(P0.05)；与正常组相比,术后1周角膜细胞数量先减少,术后1月细胞数量增多。同一手术方式术后1月细胞数量显著多于1周(P0.05)。 2.于LASIK术后1周和1月处死实验动物提取组织蛋白,采用酶联免疫吸附试验(ELISA)方法检钡(?)bFGF含量。研究发现：LASIK术后1周,角膜基质床残余30%组与对照组相比,bFGF含量显著增高(P0.05)；LASIK术后1月,角膜基质床残余30%组bFGF含量回落至正常水平,不同手术组之间无显著性差异；不同时间点同一手术方式之间比较发现,仅角膜基质床残余30%组1周和1月有显著差异(P0.05)。 3.体外采用FX-4000细胞力学柔性基底拉伸系统,对原代提取的兔角膜成纤维细胞分别进行频率为O.1Hz、拉伸幅度为5%、10%、15%的周期性机械拉伸,并于6h和24h收集细胞培养液上清,采用ELISA法检(?)(?)bFGF的含量。研究发现：拉伸6h后,15%拉伸组bFGF含量较对照组显著增高(P0.05),24h后显著性降低(P0.05)。实验结果表明：在体不同切削量的LASIK手术后角膜基质细胞的增殖发生变化,bFGF的表达主要集中在基质层。LASIK术后角膜组织及周期性拉伸作用下角膜成纤维细胞bFGF早期表达的显著上调,将有益于术后角膜组织的修复。
[Abstract]:Laser in situ keratomileusis (LASIKK) is a kind of corneal refractive surgery which is widely used in the treatment of myopia. Every year, nearly one million people are treated with LASIK. LASIK improves refractive errors mainly by changing the curvature of the central optical area of the cornea. This procedure is performed in the stromal layer below the corneal flap, which not only preserves the integrity of the epithelial layer and the anterior elastic layer of the cornea, but also prevents the occurrence of haze under the cornea. The repair process of corneal injury after LASIK is a complicated process involving many factors. Among them, the change of mechanical environment is an important factor in corneal tissue repair after LASIK. The corneal layer becomes thinner after LASIK. Under the same intraocular pressure, the stress of cornea increases obviously. The mechanical environment of corneal fibroblasts has changed, which may affect the growth and proliferation of corneal fibroblasts. Basic Fibroblast Growth FactorbFGFs can not only promote wound healing and repair of damaged tissues, but also induce the corresponding morphological changes of injured cells. Therefore, the expression and distribution of bFGF in corneal tissue and corneal fibroblasts in vitro and in vitro, and the effects of mechanical stimulation on the proliferation of rabbit corneal stromal cells were studied. To explore the role of mechanical factors in the repair of corneal injury after LASIK, and to provide theoretical basis and guidance for clinical treatment of myopia. The main contents and conclusions of this paper are as follows: 1. The animal model of LASIK operation with different cutting amount was established to observe the expression of bFGF in corneal epithelium and stromal layer by immunofluorescence staining and the proliferation of corneal stromal cells by hematoxylin-eosin) staining. It was found that the fluorescence intensity in the stromal layer was significantly higher than that in the epithelium 70% week group than that in the epithelial layer P0.05.The number of corneal cells decreased at the first week after operation, compared with the normal group, but the fluorescence intensity in the stromal layer was significantly higher than that in the control group. The number of cells increased 1 month after operation. The number of cells in one month after operation was significantly more than 1 week after operation (P 0.05). 2. After 1 week and 1 month after LASIK, the experimental animals were killed to extract the histone protein, and the content of bFGF was detected by Elisa. It was found that the content of bFGF in 30% group of residual corneal stroma bed was significantly higher than that in control group at 1 week after operation, and the content of bFGF in 30% group of residual corneal stroma bed decreased to the normal level one month after operation, but there was no significant difference between different groups. Compared with the same operation method at different time points, only the corneal stromal bed residual 30% group had significant difference in 1 week and 1 month (P 0.05). 3. In vitro, the primary rabbit corneal fibroblasts were subjected to mechanical stretching of 0.1 Hz with a tensile range of 5 1010% and 15% periodic mechanical stretching, and the supernatants were collected at 6 h and 24 h, respectively, using the FX-4000 cell mechanical flexible substrate stretching system, and the cultured rabbit corneal fibroblasts were collected at 6 h and 24 h respectively, and the cell culture medium supernatants were collected at 6 h and 24 h respectively. The content of bFGF was detected by ELISA. The results showed that the content of bFGF in the 15% stretch group was significantly higher than that in the control group after 6 hours of stretching. The results showed that the expression of bFGF in corneal stromal cells after LASIK surgery was mainly in the early stage of corneal fibroblast bFGF after stromal layer. LASIK and periodic stretching. A significant up-regulation of the expression, It will be beneficial to the repair of corneal tissue after operation.
【学位授予单位】：太原理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R779.63

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