扩张性角膜疾病的力学生物学研究

发布时间：2018-02-23 00:19

本文关键词： 角膜膨隆角膜成纤维细胞力学牵拉炎性因子基质金属蛋白酶胶原　出处：《太原理工大学》2016年博士论文　论文类型：学位论文

【摘要】：扩张性角膜疾病是需要进行角膜移植的第二大适应症,包括圆锥角膜和屈光手术后的角膜膨隆(医源性角膜膨隆)。角膜屈光手术后角膜变薄,在眼内压、用力揉眼等情况下使角膜的应变增大;同时,角膜屈光手术后上皮细胞分泌炎性因子IL-1β参与伤口愈合,尤其是术后干眼症会导致泪液中IL-1β的升高,使基质层的细胞处于复杂的力学和生物学环境中。当角膜基质层受损后,角膜基质细胞会分化为角膜成纤维细胞参与角膜的损伤修复和伤口愈合。圆锥角膜患者泪液中和上皮细胞TNF-α表达的升高,以及圆锥角膜患者常伴有异常揉眼的习惯,使得圆锥角膜成纤维细胞也处于复杂的力学和生物学环境。为了研究扩张性角膜疾病的发生机制,本文对角膜成纤维细胞在IL-1β存在的条件下施加了不同幅度的力学牵拉,研究角膜成纤维细胞形态和细胞外基质的变化。同时,提取了圆锥角膜成纤维细胞,研究炎性因子TNF-α对圆锥角膜成纤维细胞基质金属蛋白酶及其抑制剂表达的影响。主要工作及研究结果如下:(1)体外对角膜成纤维细胞施加了不同幅度(5%、10%、15%)的力学牵拉,研究力学刺激对角膜成纤维细胞形态的影响,发现力学牵拉使角膜成纤维细胞的F-actin发生收缩,牵拉幅度越大F-actin收缩程度越大,使角膜成纤维细胞的迁移受到抑制。(2)探讨了在炎性因子IL-1β存在时,不同幅度(5%、10%)、不同持续时间(12h、24h、36h)的力学牵拉作用对角膜成纤维细胞i型胶原、lumican表达的影响。同时,用明胶酶谱法和westernblot对不同幅度作用36h时角膜成纤维细胞上清液中mmp2、mmp9的表达进行了检测。研究发现:力学牵拉、IL-1β短时间(12h)单独作用能降低角膜成纤维细胞Ⅰ型胶原的合成,这可能是角膜成纤维细胞对其做出的的应急反应。低幅度(5%)牵拉单独作用较长时间(24h、36h)能促进角膜成纤维细胞Ⅰ型胶原的合成,高幅度(10%)牵拉则抑制Ⅰ型胶原的合成。IL-1β与力学牵拉共同作用使角膜成纤维细胞Ⅰ型胶原合成降低。力学牵拉短时间单独作用时,低幅度能促进lumican的表达,高幅度却抑制其表达;IL-1β与力学牵拉共同作用能促进lumican的表达。IL-1β能诱导角膜成纤维细胞mmp9的表达,并促进mmp2的表达;力学牵拉能进一步促进IL-1β引起的mmp2表达的升高。说明炎性因子与力学牵拉共同作用使角膜成纤维细胞胶原合成减少、基质降解增多,可能会引起医源性角膜膨隆的发生。(3)圆锥角膜患者泪液中TNF-α表达的升高以及上皮细胞TNF-α的高表达,使圆锥角膜成纤维细胞处于炎症环境中,通过对圆锥角膜成纤维细胞施加不同浓度的TNF-α作用,发现TNF-α能促进圆锥角膜成纤维细胞mmps的表达,而抑制其timps的表达。MMPs/TIMPs的失衡将导致圆锥角膜成纤维细胞外基质的降解增多,进而可能导致角膜组织结构的完整性遭受破坏。(4)对圆锥角膜成纤维施加TNF-α和力学牵拉的作用,研究发现两者共同作用使圆锥角膜成纤维细胞的增殖受到抑制,细胞凋亡不受影响。角膜成纤维细胞数目的减少可能会使细胞外基质的合成降低,角膜的生物力学性能发生改变。综上所述:炎性因子和力学刺激通过调节细胞外基质的代谢、角膜成纤维细胞的增殖在扩张性角膜疾病的发生中起着重要的作用。
[Abstract]:The expansion of corneal diseases is the need for corneal transplantation second indications, including keratoconus after refractive surgery and corneal ectasia (iatrogenic corneal ectasia). After corneal refractive surgery and corneal thinning in intraocular pressure, eye rubbing force under the condition that the corneal strain increases; at the same time, corneal refractive surgery after the epithelial cells secrete inflammatory factor IL-1 is involved in wound healing, especially postoperative dry eye would lead to an increase in tear IL-1 beta, the stroma cells in mechanical and biological environment in the complex. When the cornea is damaged, corneal stromal cells can differentiate into corneal fibroblasts in corneal damage and repair wound healing. Tears of patients with keratoconus and expression of epithelial cells of TNF- alpha increased, and keratoconus patients often accompanied by abnormal Rouyan habits, the keratoconus fibroblasts are complex and Mechanical Biological environment. In order to study the pathogenesis of corneal expansion diseases, the corneal fibroblasts in the presence of beta IL-1 applied mechanics of different amplitude changes of corneal stretch, morphology and cell extracellular matrix fibers. At the same time, the extraction of keratoconus fibroblasts on inflammatory factor TNF- alpha effect of expression of matrix metalloproteinase and its inhibitor of keratoconus. The main work and research results are as follows: (1) in vitro on corneal fibroblasts was applied into different ranges (5%, 10%, 15%) of the mechanical stretch of mechanical stimulation effect of fibroblast morphology on the cornea, and found that mechanical stretch. Corneal fibroblasts F-actin shrink, stretch the bigger the contraction of F-actin is greater, the corneal fibroblast migration was inhibited. (2) discussed in the presence of inflammatory factor IL-1 beta, different The amplitude (5%, 10%), different duration (12h, 24h, 36h) the mechanical stretch effect on corneal fibroblast collagen type I, lumican expression. At the same time, and Westernblot of different amplitude 36h corneal fibroblast MMP2 in the supernatant by gelatin zymography, and the expression of MMP9 was detected study found that: the mechanical stretch, IL-1 beta short time (12h) alone can reduce corneal collagen synthesis in fibroblasts, which may be the response of corneal fibroblasts to make its low amplitude. (5%) stretch alone a longer period of time (24h, 36h) can promote corneal type I collagen synthesis in fibroblasts, high amplitude (10%) led by the synthesis of.IL-1 beta and mechanical pull inhibited collagen stretch to make corneal fibroblasts collagen synthesis decreased. Mechanical stretch short time alone, low amplitude can promote the expression of lumican, high amplitude But the degree of inhibition of its expression; IL-1 beta and mechanical pull together to.IL-1 beta can expression of fibroblast induced corneal MMP9 promote the expression of lumican, and promote MMP2 expression; mechanical stretch can increase further promote IL-1 beta induced MMP2 expression. The inflammatory factor and mechanical stretch joint action of the cornea fibroblasts reduced collagen synthesis, extracellular matrix degradation increased, may cause iatrogenic corneal ectasia. (3) increased and the high expression of epithelial cells of TNF- alpha TNF- alpha expression in tears of patients with keratoconus, the cone corneal fibroblasts in the inflammatory environment, the effects of different concentrations of alpha TNF- applied by the cone corneal fibroblasts, TNF- alpha keratoconus can promote the expression of MMPs, expression of.MMPs/TIMPs and inhibit the imbalance of TIMPs will lead to keratoconus fibroblast extracellular matrix degradation. Much, which may lead to the integrity of the corneal tissue structure damage. (4) applied to a fiber TNF- alpha and mechanical stretch of keratoconus, the study found that both of them make the corneal fibroblast proliferation was inhibited, apoptosis is not affected. Corneal fibroblasts may lead to the reduction in the number of the synthesis of extracellular matrix decreased and the corneal biomechanical properties change. Conclusion: inflammatory factors and mechanical stimulation by regulating the metabolism of extracellular matrix, corneal fibroblast proliferation plays an important role in the expansion of corneal disease occurrence.

【学位授予单位】：太原理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R772.2;R318.01

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