重组人组织激肽释放酶结合蛋白对隐形眼镜护理液所致细胞损伤的修复研究

发布时间：2018-04-13 14:28

本文选题：隐形眼镜护理液 + 组织激肽释放酶结合蛋白　；参考：《华侨大学》2017年硕士论文

【摘要】：近年来,许多文献报道多功能型隐形眼镜护理液能引起眼角膜损伤。据报道,多功能型护理液能引起人角膜上皮细胞(HCE)氧化应激反应、炎症反应,造成细胞凋亡,并且多功能型护理液是通过激活NF-κB信号通路来诱发细胞炎症反应。动物实验也表明多功能型护理液引起明显的兔角膜组织损伤并降低角膜对细菌的抵抗力。人组织激肽释放酶结合蛋白(Kal,Kallistatin)是一种功能多效的蛋白,也是一种丝氨酸蛋白酶抑制剂,能参与多条细胞通路的调控,具有促进血管修复和降低高血压、炎症、氧化应激、细胞凋亡等作用,能抑制NF-κB信号通路达到抗炎的作用。与Kal同一家族的SERPINA3K对HCE细胞具有抗炎,抗血管生成和抗氧化活性。基于Kal的功能多效性与SERPINA3K对角膜上皮细胞的生物活性,我们推测Kal可能对HCE损伤有较好的修复作用。主要研究内容:(1)制备Kal蛋白:通过筛选实验室构建保存的重组人Kal(rhKal)的酵母菌株,得到高表达的菌株,利用发酵罐技术进行诱导表达,进一步分离、纯化rhKal蛋白,经SDS-PAGE检测与ELISA定量,得到的rhKal是高纯度高浓度的。(2)市售五种隐形眼镜护理液(含除过氧化氢外的杀菌成分)对HCE细胞毒性验证:使用CCK-8方法,处理时间为3 h,筛选出能使HCE细胞活力显著下降的护理液含量,再观察该含量下细胞形态、检测细胞超氧化阴离子水平及细胞凋亡水平。(3)rhKal对护理液所致HCE损伤的修复作用:将rhKal蛋白应用于五种护理液细胞损伤模型中,对其抗氧化、抗凋亡生物活性进行研究。(4)rhKal对H_2O_2所致HCE及人结膜上皮细胞(HConEpiC)损伤的修复作用:用H_2O_2对HCE和HConEpiC处理3 h构建损伤模型,考察rhKal蛋白对其损伤修复的影响。结果显示:rhKal对五种隐形眼镜护理液造成的HCE细胞损伤有显著修复作用,能减少细胞内超氧阴离子,抑制护理液引起的细胞凋亡,具有一定的剂量依赖性。rhKal对H_2O_2引起HCE和HConEpiC细胞损伤也具有显著修复作用,能有效的抑制细胞凋亡。本研究说明rhKal对隐形眼镜护理液引起的细胞损伤均具有修复作用,为研制一种针对隐形眼镜护理液损伤的滴眼修复液奠定了基础。
[Abstract]:In recent years, it has been reported that multifunctional contact lenses can cause corneal injury.It has been reported that multifunctional care fluid can induce oxidative stress, inflammatory response and apoptosis in human corneal epithelial cells (HCE), and multifunctional care fluid induces cellular inflammatory response by activating NF- 魏 B signaling pathway.Animal experiments also showed that multifunctional preservative induced obvious corneal tissue injury and decreased corneal resistance to bacteria.Human tissue kallikrein binding protein (Kallistatin) is a multifunctional protein and a serine protease inhibitor, which is involved in the regulation of multiple cellular pathways and can promote vascular repair and reduce hypertension, inflammation and oxidative stress.Apoptosis can inhibit the NF- 魏 B signaling pathway to achieve anti-inflammatory effect.SERPINA3K of the same family as Kal has anti-inflammatory, anti-angiogenesis and anti-oxidation activities on HCE cells.Based on the functional versatility of Kal and the biological activity of SERPINA3K on corneal epithelial cells, we speculate that Kal may have a better repair effect on HCE injury.The main content of this study was to prepare Kal protein: the yeast strain with high expression was obtained by screening the yeast strain of recombinant human Kalhal Kalin, and the rhKal protein was further isolated and purified by fermentor technique.By SDS-PAGE detection and ELISA quantification, the obtained rhKal is a high-purity and high concentration of .t2.) five kinds of contact lens care solution (containing bactericidal components other than hydrogen peroxide) are used to verify the cytotoxicity of HCE cells: using CCK-8 method,After treatment for 3 h, the contents of nursing fluid which could significantly decrease the viability of HCE cells were screened out, and the cell morphology was observed at the same time.To investigate the effect of superoxide anion and apoptosis on HCE damage induced by care fluid, rhKal protein was applied to five kinds of cell injury models.The anti-apoptotic biological activity was studied. The repair effect of rhKal on H_2O_2 induced HCE and human conjunctival epithelial cell (HConEpiC3) injury was studied. HCE and HConEpiC were treated with H_2O_2 for 3 h to construct the injury model, and the effect of rhKal protein on the damage repair was investigated.The results showed that the HCE cell damage induced by five kinds of contact lens solution could be significantly repaired by the solution. It could reduce the superoxide anion in the cells and inhibit the apoptosis induced by the solution.In a dose-dependent manner, rhKal can significantly repair HCE and HConEpiC cell damage induced by H_2O_2, and can effectively inhibit apoptosis.This study shows that rhKal can repair the cell damage induced by contact lens care fluid, which lays a foundation for the development of a kind of eye repair fluid for contact lens injury.
【学位授予单位】：华侨大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R772.2

【参考文献】