【作者】 于晓明；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2013， 硕士

【摘要】 研究目的角膜上皮创伤愈合延迟是目前眼科临床常见的糖尿病眼部并发症之一,治疗棘手,发生机制尚不清楚。糖基化终末产物(advanced glycation end products, AGEs)是在持续高糖条件下,蛋白质的氨基与糖的醛基在非酶催化反应下生成的终产物,与糖尿病慢性并发症的发生发展相关。AGEs促进ROS的生成,而ROS可通过不同的途径诱导细胞凋亡。因此本课题通过研究AGEs对THCEs凋亡作用及其与ROS的关系,探讨AGEs在糖尿病角膜创伤愈合延迟中的作用机制。研究方法利用牛血清白蛋白(BSA)和葡萄糖体外制备AGE-BSA及其对照物。体外培养THCE细胞与不同浓度(50μg/ml,100μg/ml,200μg/ml,400μg/ml)的AGEs修饰牛血清白蛋白(AGE-BSA)在体外共同培养不同时间(6h,12h,24h,48h)。应用Annexin V-Fitc和碘化丙啶(PI)与细胞共同孵育,通过流式细胞仪检测并定量分析细胞凋亡百分率；采用Western blot检测凋亡蛋白Bcl-2,Bax蛋白表达。选用刺激效果最好的AGE-BSA浓度和作用时间,抗氧化剂NAC (20μmol/L)预先作用1小时清除内源性ROS或NADPH氧化酶抑制剂DPI (10μM)和Apocynin (300μM)孵育1小时抑制NADPH氧化酶后,实验分为9组：空白对照组、BSA对照组、AGE-BSA组、AGE-BSA+NAC组、AGE-BSA+DPI组、AGE-BSA+Apocynin组、DPI组、Apocynin组、NAC组,应用流式细胞仪检测细胞凋亡百分率。选用刺激效果最好的AGE-BSA浓度和作用时间,用NADPH氧化酶抑制剂DPI (10μM)和Apocynin (300μM)抑制NADPH氧化酶后,实验分为5组,分别为空白对照组、BSA对照组、AGE-BSA组、AGE-BSA+DPI组、AGE-BSA+Apocynin组,用Western blot检测Bax蛋白表达。结果流式细胞仪凋亡检测分析THCEs凋亡率结果显示：200μg/ml AGE-BSA刺激细胞6h,凋亡细胞明显增加(P<0.05),24h达高峰(P<0.05),与对照组相比,差异有统计学意义； BSA对照组与空白对照组相比凋亡率改变无统计学意义。50μg/ml AGE-BSA能够明显刺激THCE细胞凋亡(P<0.05),100μg/ml,200μg/ml和400μg/ml AGE-BSA均能够显著增加角膜上皮细胞凋亡(P<0.05),与对照组比差异有统计学意义；BSA对照组与空白对照组相比凋亡率改变无统计学意义。Western-blot结果显示：200ug/ml AGE-BSA刺激THCE细胞6h,12h,24h,Bax蛋白表达增强,12h达高峰(P<0.05)；Bcl-2蛋白表达减弱,24h最明显(P<0.05)。分别用ROS清除剂NAC清除ROS,NADPH氧化酶抑制剂DPI、Apocynin抑制NADPH氧化酶从而抑制细胞内ROS的生成后,AGE-BSA200ug/ml刺激THCE细胞24h,流式结果显示,与AGE-BSA组相比,流式细胞仪检测细胞凋亡率均明显降低,差异有统计学意义(P<0.05)；与空白对照组及BSA对照组相比,差异无统计学意义。应用NADPH氧化酶抑制剂DPI、Apocynin抑伟NADPH氧化酶从而抑制细胞内ROS的生成后,AGE-BSA200ug/ml刺激THCE细胞24h,Western blot检测Bax蛋白表达降低,与AGE-BSA组相比差异有统计学意义(P<0.05)；与空白对照组及BSA对照组相比,差异无统计学意义。结论AGE-BSA诱导THCE细胞凋亡,而且可能是通过NADPH氧化酶途径生成的活性氧(ROS)进而促进Bax的表达发挥促凋亡作用的。AGEs可能是引起糖尿病角膜上皮创伤愈合延迟的重要致病因素之一。更多还原

【Abstract】 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.更多还原