晚期糖基化终末产物及其受体在糖尿病性白内障发病中的作用

发布时间：2018-01-25 09:22

本文关键词： 晚期糖基化终末产物晚期糖基化终末产物受体糖尿病性白内障　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：研究背景: 糖尿病性白内障(diabetic cataract, DC)是糖尿病并发症中仅次于视网膜病变的第二大致盲性眼病。糖尿病患者体内积聚的大量晚期糖基化终末产物(advanced glycation end-products, AGEs)同其受体(receptor for advanced glycation end-products, RAGE)结合后,能够引起细胞内活性氧簇合成增多、核因子转录活性增加等细胞反应,造成组织氧化损伤、炎症反应,是AGEs在人体内发挥作用并导致疾病的一个重要途径。目的:本课题旨在通过观察RAGE在糖尿病性白内障、年龄相关性白内障及正常晶状体上皮细胞中的表达情况,体外实验研究AGEs对于人晶状体上皮细胞氧化状态以及RAGE表达的影响,初步探讨两者相互作用在糖尿病性白内障发生发展中的作用及可能机制,为糖尿病性白内障基础研究及药物防治提供新思路和实验依据。方法: (1)收集70例糖尿病性白内障、72例年龄相关性白内障及24例正常晶状体前囊膜;收集糖尿病性白内障患者性别、年龄、糖尿病病程、糖化血红蛋白水平、白内障类型以及是否伴发糖尿病视网膜病变等6项临床资料。采用免疫组化的方法检测RAGE在糖尿病性白内障、年龄相关性白内障及正常晶状体上皮细胞上的表达;分别比较并分析RAGE在糖尿病性白内障、年龄相关性白内障及正常晶状体上皮细胞中的表达差异;使用logistic回归分析法分析糖尿病患者晶状体上皮细胞中RAGE表达同6项临床资料间的相关性。 (2)体外培养人晶状体上皮细胞系(SRA01/04),实验组分别加入浓度为0 mg/ml、0.25 mg/ml、0.5 mg/ml、1 mg/ml的AGE-BSA;对照组则分别加入相同浓度梯度的对照牛血清白蛋白(bovine serum albumin, BSA),同细胞共孵育24h后:使用ROS检测试剂盒,流式细胞技术观测各组细胞内ROS的含量;使用GSH及GSSG检测试剂盒,比色法测算各组细胞内GSSG/GSx比值;使用免疫印记法检测各组细胞中RAGE表达的情况。(3)体外培养人晶状体上皮细胞系(SRA01/04),实验组加入浓度为0.5 mg/ml的AGE-BSA;对照组加入相同浓度的对照BSA,分别观察各组加入干预或对照试剂第0、2、8、16、24h后: ROS含量的变化,GSSG/GSx比值的改变以及RAGE的表达情况。结果: (1)①糖尿病性白内障及年龄相关性白内障患者晶状体前囊膜上皮细胞均可见有RAGE不同程度的阳性着色。而透明晶状体前囊膜则未见类似着色。RAGE在糖尿病性白内障晶状体及透明晶状体上皮细胞上的表达间存在显著性差异(P0.0125);RAGE在年龄相关性白内障及透明晶状体上皮细胞上的表达间存在显著性差异(P0.0125);RAGE在糖尿病性白内障晶状体及年龄相关性白内障晶状体上皮细胞上的表达间存在显著性差异(P0.0125)。 ②临床数据中糖尿病病程和糖化血红蛋白水平,与RAGE表达情况的相关性具有统计学意义(P0.05),并且这种相关性为正相关(OR1),而其他4项包括患者性别、年龄、白内障类型以及是否伴发有糖尿病视网膜病变同RAGE表达情况的相关性则不具有统计学意义(P0.05)。 (2)①随着AGEs作用浓度的增加, ROS含量不断增加,但在对照组,细胞内ROS含量无明显变化。在干预浓度为0 mg/ml及0.25 mg/ml时,作用24 h后,实验组及对照组细胞内ROS含量无明显统计学差异(P0.05);在干预浓度为0.50 mg/ml及1.00 mg/ml时,作用24 h后,实验组及对照组ROS含量存在显著统计学差异(P0.05)。随着AGEs作用浓度的增加,细胞内GSSG/GSx也不断增加:实验组细胞内GSSG/GSx的比值,从初始干预浓度为0 mg/ml时的1.7±0.26%,增长至干预浓度为1.00 mg/m时的12.83±0.40%,而对照组则无明显变化。在浓度为0 mg/ml及0.25 mg/ml时,作用24 h后,实验组及对照组GSSG/GSx无明显统计学差异(P0.05);在浓度为0.50 mg/ml及1.00 mg/ml时,作用24 h后,实验组及对照组GSSG/GSx存在显著统计学差异(P0.05)。 ②AGEs能够诱导晶状体上皮细胞RAGE在蛋白质水平上的表达增加:随着AGEs浓度的增加,晶状体上皮细胞内RAGE蛋白表达不断增加;在AGEs浓度为0.5 mg/ml及1.00 mg/ml时,RAGE蛋白表达较对照组显著增加(P0.05)。 ③随着AGEs作用时间的增加,细胞内ROS含量不断增加,这种增加在干预后0、2、8 h时变化不明显,而在干预后第16 h及24 h时ROS的增加则较为明显。但在对照组,ROS的含量并无明显变化。在干预后第16 h及24 h时,实验组细胞内ROS含量明显高于对照组,存在明显统计学差异(P0.05)。随着AGEs作用时间的增加,细胞内GSSG/GSx也不断增加:实验组细胞内GSSG/GSx的比值,在干预后0 h时的0.63±0.15%,增长至24 h时的10.63±0.40%。但在对照组,GSSG/GSx比值未出现明显变化。在AGEs作用于细胞初始的8 h内,细胞内GSSG/GSx的比值增长迅速,而在其后的16 h内,这种增长变得较为平缓。比较相同作用时间内实验组及对照组GSSG/GSx的比值,各组间具有统计学差异(P0.05)。 ④AGEs能够诱导晶状体上皮细胞RAGE在蛋白质水平的表达增加:随着AGEs作用时间的延长,RAGE表达水平不断增高;在作用时间为16 h及24 h时,同对照组相比,表达具有统计学意义(P0.05)。结论: RAGE在糖尿病性白内障及年龄相关性白内障患者晶状体上皮细胞中高表达,在正常透明晶状体上皮细胞中几乎不表达;AGEs能够改变晶状体上皮细胞氧化状态,上调RAGE的表达;AGEs-RAGE相互作用可能在糖尿病性白内障发病中起重要作用。
[Abstract]:Research background:
Diabetic cataract (diabetic cataract DC) is a complication of diabetes retinopathy after approximately second blind eye disease in diabetes. The accumulation of a large number of advanced glycation end products (advanced glycation, end-products, AGEs) and its receptor (receptor for advanced glycation end-products, RAGE) combined, can cause the synthesis of reactive oxygen species in the cell increase, enhance the transcription activity of nuclear factor cell reaction, cause oxidative tissue damage, inflammation, AGEs play a role in the human body is an important way to cause disease. Objective: This study is designed to observe the expression of RAGE in diabetic cataract, age-related cataract and normal lens epithelial cells in vitro. Experimental Study on the influence of AGEs on oxidation state of human lens epithelial cells and the expression of RAGE, a preliminary exploration of the interaction between the two The role and possible mechanism in the development of diabetic cataract can provide new ideas and experimental basis for the basic research and drug control of diabetic cataract.
Method:
(1) collected 70 cases of diabetic cataract, 72 cases of age-related cataract and 24 cases of normal anterior capsule; diabetic cataract patients with gender, age, duration of diabetes, HbA1c levels, and whether the type of cataract associated with diabetic retinopathy and 6 clinical data. Immunohistochemical method was used to detect RAGE in diabetic cataract, expression of age-related cataract and normal lens epithelial cells; compare and analyze the RAGE expression in diabetic cataract, age-related cataract and normal lens epithelial cells; using logistic regression analysis to analyze the expression of RAGE correlated with the 6 clinical data between the epithelial cells of diabetic patients in the lens.
(2) human lens epithelial cells cultured in vitro (SRA01/04), the experimental group were added at a concentration of 0 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml AGE-BSA; the control of bovine serum albumin in control group were cultured in the same concentration gradient (bovine serum, albumin, BSA), after 24h cells were incubated with: ROS kit and flow cytometry were observed in ROS cells; using GSH and GSSG test kit, colorimetric method to measure the intracellular GSSG/GSx ratio; using Western blot to detect the expression of RAGE of cells. (3) human lens epithelial cells cultured in vitro (SRA01/04), experimental group with a concentration of 0.5 mg/ml AGE-BSA; control group BSA with the same concentration, were observed with the 0,2,8,16,24h after the intervention or control reagent: the changes of ROS content, GSSG/GSx ratio change and the expression of RAGE.
Result:
(1) of diabetic cataract and age-related cataract lens anterior capsule epithelial cells were observed RAGE positive staining in different degrees. There is a significant difference between the expression of transparent lens capsule coloring.RAGE in diabetic cataract lens and lens epithelial cells (P0.0125); the significant the differential expression of RAGE in age-related cataract and transparent lens epithelial cells on the (P0.0125); there was a significant difference between the expression of RAGE in diabetic cataract and age-related cataract lens epithelial cells on the (P0.0125).
The clinical data of diabetes and HbA1c levels, and the correlation between the expression of RAGE was statistically significant (P0.05), and the correlation between positive correlation (OR1), while the other 4 patients including gender, age, type of cataract and whether there is associated with diabetic retinopathy with the expression of RAGE was not statistically meaning (P0.05).
(2) with the increase of AGEs concentration, ROS content increased, but in the control group, the content of ROS in cells. No significant changes in the intervention concentration of 0 mg/ml and 0.25 mg/ml, 24 h, the experimental group and the control group the content of ROS cells had no significant difference (P0.05) in the intervention; the concentration of 0.50 mg/ml and 1 mg/ml, 24 h, the experimental group and the control group ROS. There was significant difference (P0.05). With the increase of the concentration of AGEs, intracellular GSSG/GSx increased ratio of experimental group GSSG/GSx cells, from the initial dry pre concentration was 0 mg/ml 1.7 + 0.26% to increase, intervention concentration was 1 mg/m 12.83 + 0.40%, while the control group had no obvious change. At the concentration of 0 mg/ml and 0.25 mg/ml, 24 h, experimental group and control group GSSG/GSx had no significant difference (P0.05); at the concentration of 0.50 mg/ml and 1 mg/ml, as After 24 h, there were significant differences in GSSG/GSx between the experimental group and the control group (P0.05).
The increased expression of AGEs can induce RAGE lens epithelial cells at the protein level, with the increase of AGEs concentration, the expression of RAGE protein in lens epithelial cells increased; when the concentration of AGEs was 0.5 mg/ml and 1 mg/ml, RAGE protein expression increased significantly compared with the control group (P0.05).
With the increase of AGEs time, intracellular ROS content increased, the increase in 0,2,8 after the intervention of h did not change significantly, but after the intervention of sixteenth h and 24 h ROS increased obviously. But in the control group, no significant changes in the content of ROS. After the intervention of sixteenth h and 24 h when the content of ROS in experimental group cells was significantly higher than control group, there was statistically significant difference (P0.05). With the increase of AGEs. The intracellular GSSG/GSx increased ratio of experimental group GSSG/GSx cells, 0.63 + 0.15% at 0 h after the intervention, increased to 24 when h 10.63 + 0.40%. but in the control group, the GSSG/GSx ratio did not change significantly. The effect of AGEs on the cell in the initial 8 h, the ratio of increase of intracellular GSSG/GSx quickly, and then the 16 h, the growth became slow. The same time the experimental group and control group than GSSG/GSx There was a statistical difference between each group (P0.05).
(4) AGEs can induce increased expression of RAGE in the protein level of lens epithelial cells: with the prolongation of AGEs action time, the level of RAGE expression is increasing. When the action time is 16 h and 24 h, the expression is statistically significant compared with the control group (P0.05).
Conclusion:
The high expression of RAGE in lens epithelial cells in cataract patients with diabetic cataract and age-related, almost no expression in normal lens epithelial cells; AGEs can change the redox state of LECs, upregulating the expression of RAGE; AGEs-RAGE interaction may play an important role in the pathogenesis of diabetic cataract.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R587.1;R776.1

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