Toll样受体4及相关炎性细胞因子在糖尿病大鼠视网膜中的表达

Expression of Toll-like receptor 4 and inflammatory factors in retina of diabetic rats

目的 观察糖尿病大鼠视网膜中Toll受体4（TLR4）、炎性细胞因子的表达水平以及视网膜中白细胞聚集与视网膜通透性改变。方法 Brown Norway大鼠120只，剔除自然死亡14只，随机分为实验组和对照组，每组均为53只大鼠。实验组大鼠腹腔注射链脲佐菌素建立糖尿病模型；对照组大鼠腹腔注射等体积柠檬酸-柠檬酸钠缓冲液。建模后4周行定量聚合酶链反应、蛋白质免疫印迹法检测，观察大鼠视网膜中TLR4的基因及蛋白表达，酶联免疫吸附试验测定大鼠视网膜匀浆上清液中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、单核细胞趋化蛋白-1（MCP-1）等炎性细胞水平；吖啶橙眼底血管造影观察视网膜中白细胞密度；伊凡思蓝（EB）检测视网膜通透性。结果 对照组、实验组大鼠视网膜TLR4 mRNA、蛋白表达量比较，差异均有统计学意义（F=1.606、0.789，P＜0.05）；视网膜匀浆上清液中MCP-1、IL-1β、TNF-α表达量比较，差异均有统计学意义（F=24.622、5.758、4.829，P＜0.05）。实验组、对照组大鼠视网膜中白细胞密度分别为（6.2±0.5）×10^-5、（2.2 ±0.3）×10^-5个细胞 /像素2，实验组大鼠视网膜中白细胞密度较对照组显著增加，差异有统计学意义（F=2.025，P＜0.05）。实验组、对照组大鼠视网膜EB渗漏量分别为（23.41±4.47）、（13.22±3.59） ng/mg，实验组大鼠视网膜EB渗漏量较对照组增加，差异有统计学意义（F=21.08，P＜0.05）。结论 糖尿病大鼠视网膜中TLR4及炎性细胞因子表达均显著提高；视网膜中白细胞密度和视网膜渗漏量增加。

Objective To observe the expression of Toll-like receptor 4 （TLR4） and inflammatory cytokines, leucocytic density and permeability in retina of diabetic rat. Methods A total of 106 Brown Norway rats were randomly divided into experimental group and control group with 53 rats in each group. Diabetic model was established in experimental group by intraperitoneal injection of streptozotocin, and control rats received intraperitoneal injection of an equal volume of citric acid-sodium citrate buffer. Four weeks later, the retinas were collected for further analysis. TLR4 RNA and protein expression were measured by quantitative polymerase chain reaction and Western blot. Inflammatory cytokines, including tumor necrosis factor-α （TNF-α）, interleukin-1β （IL-1β）, monocyte chemo-attractant protein-1 （MCP-1）, were measured by enzymelinked immunosorbent assay in rat retina homogenate. Leukocyte density in the retina was measured by acridine orange fundus angiography. The retinal permeability was evaluated by Evans blue （EB） staining. Results TLR4 expression was significantly increased in diabetic rats of experimental group compared with non-diabetic rats of control group （F=1.606, 0.789;P〈0.05）. Inflammatory cytokines （TNF-α, IL-1β and MCP-1） were significantly increased in retina of diabetic rats of experimental group versus non-diabetic rat of control group （F=24.622, 5.758, 4.829;P〈0.05）. The retinal leukocyte density was （6.2±0.5） ×10^-5, （2.2±0.3） ×10^-5cells / pixel2 in experimental and control group respectively, the difference was statistically significant （F=2.025, P〈0.05）. The amount of retinal EB leakage was （23.41±4.47）, （13.22±3.59） ng/mg in experimental and control group respectively, the difference was statistically significant （F=21.08, P〈0.05）. Conclusion TLR4 and inflammatory cytokines expression, leucocytic density and permeability increased significantly in retina of diabetic rat.