高糖对视网膜色素上皮细胞TLR2表达的影响

发布时间：2018-05-19 23:02

本文选题：ARPE-19细胞 + 糖尿病视网膜疾病　；参考：《重庆医科大学》2012年硕士论文

【摘要】：目的：观察高糖对体外培养的ARPE-19细胞TLR2表达的影响及机制。（1）利用RealtimePCR技术探讨TLR2mRNA在不同D-葡萄糖浓度下培养人视网膜色素上皮细胞（retinalpigmentepithelialcell,RPE）中的表达。（2）利用Westernblot技术探讨不同D-葡萄糖浓度和PKC-α抑制剂对RPE细胞TLR2蛋白和PKC-α从细胞浆向细胞膜转位情况的影响。（3）初步探讨TLR2与糖尿病视网膜病变（diabetesretinopathy，DR）的相关性，高糖对ARPE-19细胞TLR2表达的影响以及机制，为相关疾病的诊断治疗提供新的思路。方法：1.培养ARPE-19细胞。2.取第16～20代人RPE细胞用于实验，，各组ARPE-19（RPE细胞系）细胞分别刺激48h，正常糖浓度组（5.5mmol/LD-葡萄糖）、中度高糖组（15.5mmol/LD-葡萄糖)、高糖组（25.5mmol/LD-葡萄糖)、PKC-α抑制剂组（25.5mmol/LD-葡萄糖加10mmol/LGo6976PKC-α抑制剂）、高渗对照组（5.5mmol/LD-葡萄糖加20mmol/L甘露醇）。3.Real-TimePCR分析各组ARPE-19细胞TLR2的mRNA表达水平。4.WesternBlot检测各组ARPE-19细胞TLR2蛋白表达水平，以及PKC-α蛋白胞浆向胞膜的转位情况。结果：与正常糖浓度组ARPE-19细胞相比，高糖组ARPE-19细胞和中度高糖组ARPE-19细胞TLR2的mRNA和蛋白表达，以及PKC-α从细胞浆向细胞膜的转位均增加（P0.05），并且高糖组ARPE-19细胞高于中度高糖组ARPE-19细胞（P0.05）。与高糖组ARPE-19细胞比较，PKC-α抑制剂组ARPE-19细胞的TLR2蛋白表达降低（P0.05）,同时PKC-α蛋白从细胞膜向细胞浆转位情况显著降低（P0.01）。结论：1.人ARPE-19细胞有TLR2蛋白表达。2.高糖可以上调ARPE-19细胞TLR2的表达，并且随着糖浓度的升高TLR2表达也升高。3.PKC-α抑制剂可以部分拮抗高糖上调ARPE-19细胞TLR2表达的作用。4.高糖可能是通过增加PKC-α从细胞浆向细胞膜转位，激活PKC-α信号通路，来上调TLR2的表达。TLR2信号通路可能参与糖尿病视网膜病变的发生发展过程，为研究疾病的诊治提供新的思路。
[Abstract]:Objective: to observe the effect of high glucose on the expression of TLR2 in cultured ARPE-19 cells and its mechanism. (1) to investigate the expression of TLR2mRNA in cultured human retinal pigment epithelial cells (RPE) by using RealtimePCR technique. With the effect of D- glucose concentration and PKC- 伪 inhibitor on the translocation of TLR2 protein and PKC- 伪 from cytoplasm to cell membrane in RPE cells, the relationship between TLR2 and diabetic retinopathy was studied. The effect of high glucose on the expression of TLR2 in ARPE-19 cells and its mechanism provide new ideas for the diagnosis and treatment of related diseases. Method 1: 1. Culture of ARPE-19 cells. Human RPE cells of the 16th ~ (th) ~ (th) passage for solid use Test, ARPE-19(RPE cells were stimulated for 48h, normal glucose concentration group was 5.5 mmol / L LD- glucose group, moderate high glucose group was 15.5 mmol / L LD- glucose group, high glucose group was 25.5 mmol / L LD- 伪 inhibitor group and 10 mmol / L LD- 伪 PKC- 伪 inhibitor, and hypertonic control group was 5.5 mmol LD- glucose + 20mmol/L mannose. The mRNA expression level of TLR2 in ARPE-19 cells was analyzed by Real-Time PCR. 4. Western Blot was used to detect the expression of TLR2 protein in ARPE-19 cells. And the translocation of PKC- 伪 protein from cytoplasm to membrane. Results: compared with the normal glucose concentration group, the ARPE-19 cells and the ARPE-19 cells in the high glucose concentration group were higher than those in the normal glucose concentration group. The expression of mRNA and protein in ARPE-19 cells and the translocation of PKC- 伪 from cytoplasm to cell membrane increased in moderate high glucose group, and the ARPE-19 cells in high glucose group were higher than ARPE-19 cells in medium high glucose group. Compared with the high glucose group, the expression of TLR2 protein in the ARPE-19 cells decreased significantly, and the translocation of PKC- 伪 protein from the cell membrane to the cytoplasm decreased significantly. Conclusion 1. TLR2 protein was expressed in human ARPE-19 cells. High glucose could up-regulate the expression of TLR2 in ARPE-19 cells, and the expression of TLR2 increased with the increase of glucose concentration. 3. PKC- 伪 inhibitor partly antagonized the up-regulation of TLR2 expression in ARPE-19 cells by high glucose. High glucose may increase the translocation of PKC- 伪 from cytoplasm to cell membrane and activate PKC- 伪 signal pathway to up-regulate the expression of TLR2. TLR2 signaling pathway may participate in the development of diabetic retinopathy and provide a new idea for the diagnosis and treatment of diabetic retinopathy.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R774.1

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