本文關鍵詞：糖基化終末產物對人角膜上皮細胞凋亡的影響，由筆耕文化傳播整理發(fā)布。

        研究目的角膜上皮創(chuàng)傷愈合延遲是目前眼科臨床常見的糖尿病眼部并發(fā)癥之一,治療棘手,發(fā)生機制尚不清楚。糖基化終末產物(advanced glycation end products, AGEs)是在持續(xù)高糖條件下,蛋白質的氨基與糖的醛基在非酶催化反應下生成的終產物,與糖尿病慢性并發(fā)癥的發(fā)生發(fā)展相關。AGEs促進ROS的生成,而ROS可通過不同的途徑誘導細胞凋亡。因此本課題通過研究AGEs對THCEs凋亡作用及其與ROS的關系,探討AGEs在糖尿病角膜創(chuàng)傷愈合延遲中的作用機制。研究方法利用牛血清白蛋白(BSA)和葡萄糖體外制備AGE-BSA及其對照物。體外培養(yǎng)THCE細胞與不同濃度(50μg/ml,100μg/ml,200μg/ml,400μg/ml)的AGEs修飾牛血清白蛋白(AGE-BSA)在體外共同培養(yǎng)不同時間(6h,12h,24h,48h)。應用Annexin V-Fitc和碘化丙啶(PI)與細胞共同孵育,通過流式細胞儀檢測并定量分析細胞凋亡百分率；采用Western blot檢測凋亡蛋白Bcl-2,Bax蛋白表達。選用刺激效果最好的AGE-BSA濃度和作用時間,抗氧化劑NAC (20μmol/L)預先作用1小時清除內源性ROS或NADPH氧化酶抑制劑DPI (10μM)和Apocynin (300μM)孵育1小時抑制NADPH氧化酶后,實驗分為9組：空白對照組、BSA對照組、AGE-BSA組、AGE-BSA+NAC組、AGE-BSA+DPI組、AGE-BSA+Apocynin組、DPI組、Apocynin組、NAC組,應用流式細胞儀檢測細胞凋亡百分率。選用刺激效果最好的AGE-BSA濃度和作用時間,用NADPH氧化酶抑制劑DPI (10μM)和Apocynin (300μM)抑制NADPH氧化酶后,實驗分為5組,分別為空白對照組、BSA對照組、AGE-BSA組、AGE-BSA+DPI組、AGE-BSA+Apocynin組,用Western blot檢測Bax蛋白表達。結果流式細胞儀凋亡檢測分析THCEs凋亡率結果顯示：200μg/ml AGE-BSA刺激細胞6h,凋亡細胞明顯增加(P<0.05),24h達高峰(P<0.05),與對照組相比,差異有統(tǒng)計學意義； BSA對照組與空白對照組相比凋亡率改變無統(tǒng)計學意義。50μg/ml AGE-BSA能夠明顯刺激THCE細胞凋亡(P<0.05),100μg/ml,200μg/ml和400μg/ml AGE-BSA均能夠顯著增加角膜上皮細胞凋亡(P<0.05),與對照組比差異有統(tǒng)計學意義；BSA對照組與空白對照組相比凋亡率改變無統(tǒng)計學意義。Western-blot結果顯示：200ug/ml AGE-BSA刺激THCE細胞6h,12h,24h,Bax蛋白表達增強,12h達高峰(P<0.05)；Bcl-2蛋白表達減弱,24h最明顯(P<0.05)。分別用ROS清除劑NAC清除ROS,NADPH氧化酶抑制劑DPI、Apocynin抑制NADPH氧化酶從而抑制細胞內ROS的生成后,AGE-BSA200ug/ml刺激THCE細胞24h,流式結果顯示,與AGE-BSA組相比,流式細胞儀檢測細胞凋亡率均明顯降低,差異有統(tǒng)計學意義(P<0.05)；與空白對照組及BSA對照組相比,差異無統(tǒng)計學意義。應用NADPH氧化酶抑制劑DPI、Apocynin抑偉NADPH氧化酶從而抑制細胞內ROS的生成后,AGE-BSA200ug/ml刺激THCE細胞24h,Western blot檢測Bax蛋白表達降低,與AGE-BSA組相比差異有統(tǒng)計學意義(P<0.05)；與空白對照組及BSA對照組相比,差異無統(tǒng)計學意義。結論AGE-BSA誘導THCE細胞凋亡,而且可能是通過NADPH氧化酶途徑生成的活性氧(ROS)進而促進Bax的表達發(fā)揮促凋亡作用的。AGEs可能是引起糖尿病角膜上皮創(chuàng)傷愈合延遲的重要致病因素之一。

    Objective:Delayed wound healing in diabetic cornea result in significant morbidity, prolonged hospitalization, and enormous health care expenses. Advanced glycation end products (AGEs) is the final product produced in the non-enzymatic catalysis reaction in the condition of continuous glucose, and plays a critical role in the progression of chronic complications in diabetic mellitus. Thus, we investigated the effect of AGEs on THCEs apoptosis in vitro, and the relationship with ROS to study the influence and mechanism of AGEs in delayed corneal wound healing due to diabetes.Methods:tolerated human corneal epithelial cells(THCE) were cultured in vitro with AGE-BSA of the concentrations of50,100,200,400ug/ml for6,12,24,48hours. Cells were stained with annexin V-Fitc and propidium iodide(PI). Flow cytometry was used to calculate the annexin V Fitc positive cells (early stage apoptotic cells) and Annexin V Fitc/PI positive cells(late apoptotic cells). Western blot were used to detect the expression of proapoptotic protein Bcl-2and Bax. After antioxidant NAC(20μmol/L)were used for1hour or DPI (10μM) or apocrynin300μM) were used for1hour, THCE were cultured with AGE-BSA of the best concentration and time. Flow cytometry was used to calculate the ratio of apoptosis. After DPI or apocrynin were used, Western blot were used to detect the expression of proapoptotic protein Bax. Results:Flow cytometry showed that the apoptotic rates in THCE cultured with50,100or200ug/ml AGE-BSA for6,12,24or48hours were significantly higher than those in control group(P＜0.05). The apoptotic rates in200ug/ml group for24hours were significantly higher than that in100ug/ml group(P＜0.05). The apoptotic rate increased along with the increase of culture time and concentration of AGE-BSA. Western blot showed200ug/ml AGE-BSA significantly increase Bax expression and suppressed the expression of Bcl-2in a time-dependent manner with the maximal effect by24h(P＜0.05), compared with control treatment. Pretreatment with DPI, apocrynin or NAC significantly decreased the apoptotic rate compared with the only AGE-BSA group(P＜0.05). Pretreatment with DPI or apocrynin dramatically inhibited AGE-induced expression of Bax (all P＜0.05).Conclusion:AGE-BSA may promote the apoptosis of THCE by the way of ROS which is generated by NADPH oxidase. AGE modification-induced pathobiological cascade may be involved in delayed corneal wound healing due to diabetes.

糖基化終末產物對人角膜上皮細胞凋亡的影響

目錄4-5CONTENTS5-6中文摘要6-8ABSTRACT8-9符號說明10-11前言11-14材料與方法14-25結果25-27討論27-30結論30-31參考文獻31-36附圖36-41文獻綜述41-51    參考文獻45-51致謝51-52攻讀學位期間發(fā)表的學術論文52攻讀學位期間參與的科研課通52-53學位論文評閱及答辯情況表53

  本文關鍵詞：糖基化終末產物對人角膜上皮細胞凋亡的影響，，由筆耕文化傳播整理發(fā)布。