机械牵拉对角膜成纤维细胞增殖、迁移及基质代谢影响的体外实验研究

发布时间：2018-05-30 05:58

本文选题：角膜成纤维细胞 + 机械牵拉　；参考：《太原理工大学》2014年博士论文

【摘要】：继发性角膜扩张是LASIK术后最严重的并发症之一,部分病例最终不得不进行角膜移植,目前病因尚不清楚。LASIK术后角膜的应力进行了重新分布,同时损伤引起各种细胞因子和生长因子释放,基质细胞生存的微环境发生了变化。因此,本文通过对体外培养的兔角膜成纤维细胞进行力学加载,着重探讨周期性机械牵拉、细胞因子及炎性因子对角膜成纤维细胞增殖、迁移及基质代谢的影响,从力学生物学角度对LASIK术后角膜损伤修复及角膜扩张病变发生和发展的可能机理进行深入研究。主要工作及研究结果如下： (1)体外模拟LASIK术后角膜力学环境变化,探讨5%周期性牵拉条件下,表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)对角膜成纤维细胞增殖、迁移的影响。结果发现5%周期性牵拉能够促进角膜成纤维细胞增殖,但抑制了细胞迁移行为。EGF和bFGF可以减弱牵拉对细胞迁移的抑制效应。 (2)探讨了不同牵拉幅度的机械应力对角膜成纤维细胞基质金属蛋白酶(MMPs)及其抑制因子(TIMPs)、Ⅰ型胶原(Collagen Ⅰ)表达的影响。研究发现：不同幅度周期性牵拉对MMPs、TIMPs和Collagen Ⅰ表达的调节具有双向性：低幅度牵拉(5%)抑制MMP-2表达,对MT1-MMP无显著影响,同时促进TIMPs和Collagen Ⅰ表达；高幅度牵拉(15%)则促进MMP-2和MT1-MMP表达,抑制TIMPs和Collagen Ⅰ表达。实验结果表明机械牵拉在角膜成纤维细胞基质代谢调控中发挥了重要作用。 (3)LASIK术后的角膜损伤修复及圆锥角膜常常伴有炎症的发生,加之应力状态的改变,使角膜处于复杂的环境中,这些因素对角膜成纤维细胞基质代谢的影响尚不清楚。因此,我们在损伤性牵拉的条件下添加了炎性因子IL-1p,探讨炎性因子与机械牵拉联合作用对角膜成纤维细胞MMPs、TIMPs及Collagen Ⅰ表达的影响。研究发现：IL-1p与15%周期性牵拉联合作用对角膜成纤维细胞MMP-1、MMP-3和MMP-9表达有协同作用,但对MMP-2无显著影响TIMP-1显著下调,TIMP-2无显著变化；Collagen IalmRNA表达水平进一步下降,两者具有协同作用。提示炎症等因素启动了一些MMPs表达后,损伤性周期性牵拉对IL-1β诱导MMPs有放大作用,这将进一步增加角膜基质降解的风险。 (4)ERK1/2信号通路参与了损伤性机械牵拉介导的角膜成纤维细胞MMP-2的表达调控。我们首先通过MAPK家族信号通路抑制剂筛选了与15%周期性牵拉介导的MMP-2表达有关的主要信号通路。实验结果发现PD98059(MEK信号通路抑制剂)能够显著抑制机械牵拉引起的MMP-2表达,而SP600125(JNK信号通路抑制剂)和SB203850(p38信号通路抑制剂)则对MMP-2的表达没有显著影响。其次,15%周期性牵拉使ERK1/2磷酸化水平明显增强,PD98059则使ERK磷酸化水平受到明显抑制。说明ERK1/2参与了损伤性牵拉引起的MMP-2表达的调控。综合以上结论我们得出：角膜成纤维细胞能够通过调节细胞的增殖、迁移和基质代谢,对不同的机械牵拉幅度做出响应。低幅度周期性牵拉(5%)有利于角膜基质合成,高幅度周期性牵拉(15%)则可能导致角膜基质降解,尤其在炎性因子存在的情况下。
[Abstract]:Secondary corneal dilatation is one of the most serious complications after LASIK, and some cases eventually have to undergo corneal transplantation. At present, it is not clear that the stress of the cornea is redistributed after.LASIK, and the damage causes the release of various cytokines and growth factors, and the microenvironment of the survival of the stromal cells has changed. The effects of periodic mechanical pull, cytokines and inflammatory factors on the proliferation, migration and matrix metabolism of corneal fibroblasts were investigated by mechanical loading of rabbit corneal fibroblasts cultured in vitro. The possible mechanism of corneal injury repair and development and development of corneal dilatation after LASIK was studied from the mechanical and biological point of view. Carry out an in-depth study.
The main work and research results are as follows:
(1) the changes of corneal mechanical environment after LASIK were simulated in vitro. The effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on the proliferation and migration of corneal fibroblasts were investigated under 5% periodic stretch conditions. The results showed that 5% periodic traction could promote the proliferation of corneal fibroblasts, but inhibited the cell migration behavior of.EGF. And bFGF can attenuate the inhibitory effect of stretch on cell migration.
(2) the effects of mechanical stress on the expression of matrix metalloproteinase (MMPs) and its inhibitory factor (TIMPs) and type I collagen (Collagen I) in corneal fibroblasts were investigated.
The study found that the regulation of MMPs, TIMPs and Collagen I expression of different amplitude periodicity is bidirectional: low amplitude traction (5%) inhibits MMP-2 expression, has no significant effect on MT1-MMP, and promotes the expression of TIMPs and Collagen I; high amplitude traction (15%) promotes the expression of MMP-2 and MT1-MMP, and inhibits TIMPs and Collagen I expression. The results showed that mechanical traction played an important role in the regulation of matrix metabolism in corneal fibroblasts.
(3) the repair of corneal injury after LASIK and the occurrence of keratoconus often accompanied by inflammation, and the change of stress state, the cornea is in a complex environment, and the influence of these factors on the matrix metabolism of corneal fibroblasts is not clear. Therefore, we add the inflammatory factor IL-1p to the inflammatory factors under the condition of damaged traction. The effect of combined mechanical traction on the expression of MMPs, TIMPs and Collagen I in corneal fibroblasts.
It was found that the combined effect of IL-1p and 15% periodic traction had synergistic effect on the expression of MMP-1, MMP-3 and MMP-9 in corneal fibroblasts, but there was no significant effect on TIMP-1, TIMP-2 had no significant change in MMP-2, and the IalmRNA expression level of Collagen decreased further, and both had synergism. Some MMPs tables were initiated by the inflammatory factors. After injury, traumatic periodic stretch has an amplification effect on IL-1 beta induced MMPs, which will further increase the risk of corneal matrix degradation.
(4) ERK1/2 signaling pathway is involved in the regulation of MMP-2 expression in injured mechanical dragging corneal fibroblasts.
We first screened the main signaling pathways associated with the 15% periodic traction mediated MMP-2 expression through the MAPK family signal pathway inhibitors. The results showed that the PD98059 (MEK signaling pathway inhibitor) could significantly inhibit the MMP-2 expression induced by mechanical pull, while SP600125 (JNK signaling pathway inhibitor) and SB203850 (p38 signaling pathway inhibition) However, there was no significant effect on the expression of MMP-2. Secondly, 15% periodic traction increased the level of phosphorylation of ERK1/2 significantly, while PD98059 significantly inhibited the level of ERK phosphorylation. It indicated that ERK1/2 was involved in the regulation of MMP-2 expression induced by damaged traction.
We conclude that corneal fibroblasts can respond to different mechanical pull ranges by regulating cell proliferation, migration and matrix metabolism. Low amplitude periodic traction (5%) is beneficial to corneal stroma synthesis, and high periodic stretch (15%) may lead to corneal stroma degradation, especially in the presence of inflammatory factors. Under the circumstances.
【学位授予单位】：太原理工大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R772.2

【参考文献】