本文選題：隱形眼鏡護理液 + 組織激肽釋放酶結(jié)合蛋白��； 參考：《華僑大學(xué)》2017年碩士論文

【摘要】：近年來,許多文獻報道多功能型隱形眼鏡護理液能引起眼角膜損傷。據(jù)報道,多功能型護理液能引起人角膜上皮細胞(HCE)氧化應(yīng)激反應(yīng)、炎癥反應(yīng),造成細胞凋亡,并且多功能型護理液是通過激活NF-κB信號通路來誘發(fā)細胞炎癥反應(yīng)。動物實驗也表明多功能型護理液引起明顯的兔角膜組織損傷并降低角膜對細菌的抵抗力。人組織激肽釋放酶結(jié)合蛋白(Kal,Kallistatin)是一種功能多效的蛋白,也是一種絲氨酸蛋白酶抑制劑,能參與多條細胞通路的調(diào)控,具有促進血管修復(fù)和降低高血壓、炎癥、氧化應(yīng)激、細胞凋亡等作用,能抑制NF-κB信號通路達到抗炎的作用。與Kal同一家族的SERPINA3K對HCE細胞具有抗炎,抗血管生成和抗氧化活性�；贙al的功能多效性與SERPINA3K對角膜上皮細胞的生物活性,我們推測Kal可能對HCE損傷有較好的修復(fù)作用。主要研究內(nèi)容:(1)制備Kal蛋白:通過篩選實驗室構(gòu)建保存的重組人Kal(rhKal)的酵母菌株,得到高表達的菌株,利用發(fā)酵罐技術(shù)進行誘導(dǎo)表達,進一步分離、純化rhKal蛋白,經(jīng)SDS-PAGE檢測與ELISA定量,得到的rhKal是高純度高濃度的。(2)市售五種隱形眼鏡護理液(含除過氧化氫外的殺菌成分)對HCE細胞毒性驗證:使用CCK-8方法,處理時間為3 h,篩選出能使HCE細胞活力顯著下降的護理液含量,再觀察該含量下細胞形態(tài)、檢測細胞超氧化陰離子水平及細胞凋亡水平。(3)rhKal對護理液所致HCE損傷的修復(fù)作用:將rhKal蛋白應(yīng)用于五種護理液細胞損傷模型中,對其抗氧化、抗凋亡生物活性進行研究。(4)rhKal對H_2O_2所致HCE及人結(jié)膜上皮細胞(HConEpiC)損傷的修復(fù)作用:用H_2O_2對HCE和HConEpiC處理3 h構(gòu)建損傷模型,考察rhKal蛋白對其損傷修復(fù)的影響。結(jié)果顯示:rhKal對五種隱形眼鏡護理液造成的HCE細胞損傷有顯著修復(fù)作用,能減少細胞內(nèi)超氧陰離子,抑制護理液引起的細胞凋亡,具有一定的劑量依賴性。rhKal對H_2O_2引起HCE和HConEpiC細胞損傷也具有顯著修復(fù)作用,能有效的抑制細胞凋亡。本研究說明rhKal對隱形眼鏡護理液引起的細胞損傷均具有修復(fù)作用,為研制一種針對隱形眼鏡護理液損傷的滴眼修復(fù)液奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：華僑大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R772.2

【參考文獻】

相關(guān)期刊論文 前5條

1 劉榮付;;隱形眼鏡護理有學(xué)問[J];食品與健康;2013年10期

2 孫峰;楊紹斌;;關(guān)于隱形眼鏡對眼睛傷害的研究[J];實驗科學(xué)與技術(shù);2012年01期

3 Kin-Wah Chu;Peter WC Fung;Nagy Habib;Farzin Farzaneh;;Inhibition of angiogenesis and HCT-116 xenograft tumor growth in mice by kallistatin[J];World Journal of Gastroenterology;2007年34期

4 王曉來;;隱形眼鏡的佩戴與護理[J];科學(xué)24小時;2007年Z1期

5 陳煊;隱形眼鏡的產(chǎn)生、發(fā)展與趨勢[J];醫(yī)療保健器具;2005年05期

相關(guān)重要報紙文章 前1條

1 楊昭普;林聰聰;;隱形眼鏡的“隱形”傷害[N];溫州日報;2006年

，

本文編號：1744942