TLR-2、TLR-4与大鼠角膜碱烧伤早期炎症反应相关性研究

Correlation studies between TLR-2,TLR-4 and inflammatory response of rat corneal alkali burn

目的动态观察大鼠角膜碱烧伤后Toll样受体-2(Toll like-receptor 2,TLR-2)、Toll样受体-4(Toll like-receptor 4,TLR-4)与炎性因子白细胞介素-1β(interleukin-1,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达情况及变化规律,分析Toll样受体家族(Toll like-receptor familys,TLRs)与炎性因子IL-1β、TNF-α的表达是否存在相关关系。方法选用健康SPF级SD大鼠40只,右眼为烧伤实验眼,左眼为正常对照眼,用浓度为1 mol·L-1Na OH建立Ⅲ级角膜碱烧伤模型(模型建立时及模型建立后出现大鼠角膜烧伤程度不是Ⅲ级、大鼠角膜穿孔或前房积血等情况,均予以剔除,最后随机选取32只符合条件的大鼠进行后续实验),随机分为碱烧伤后3d组、7d组、14d组、21d组,每组8眼。分别在模型制作后观察大鼠眼前节,评估其角膜炎症指数,然后摘取大鼠眼球,行HE染色,计算炎症细胞数,同时用Western blot蛋白检测法检测大鼠角膜中TLR-2、TLR-4、IL-1β、TNF-α表达情况,分析各组差异,评估TLRs与炎性因子的相关性。结果在正常大鼠角膜中可检测到TLR-2、TLR-4、IL-1β、TNF-α蛋白少量表达,碱烧伤后其表达量增加,并于碱烧伤后7d达到峰值,以后逐渐递减,各组间(3d组、7d组、14d组、21d组)比较差异均有统计学意义(均为P<0.05)。相关性分析:TLR-2与IL-1β(r=0.986,P<0.05)、TNF-α(r=0.986,P<0.05)的表达量呈正相关,TLR-4与IL-1β(r=0.975,P<0.05)、TNF-α(r=0.990,P<0.05)的表达量亦呈正相关。结论TLRs参与并可能启动角膜碱烧伤的免疫炎症反应,介导炎性因子的产生。

Objective To dynamically observe the expressions of Toll like-receptor 2 （TLR-2）,TLR-4 and inflammatory cytokine IL-1 beta and TNF alpha,and analyze the correlation between TLR-2,TLR-4 and inflammatory response on rat cornea alkali burn.Methods Forty SPF healthy SD rats were excluded the anterior segment disease,the right eye was set as burn experiment and the left eye as normal control,III level corneal alkali burn model was established with concentration of 1 mol·L-1 NaOH（At the establishment and after the establishment of the model,the inconsistent degree of corneal burns,the corneal perforation and hyphema in rats,etc.,were removed,and finally randomly selected 32 eligible rats）,then were randomly grouped into 3 days,7 days,14 days and 21 days group,8 eyes in each group.The rats were observed and photographed anterior segment of each group,evaluated the corneal inflammation index,then removed the eyeball of the rats in the corresponding time points.The eyeballs were made into pathological tissue section and stained with HE method.The number of inflammatory cells in the cornea was calculated,and the expression of TLR-2,TLR-4,IL-1 beta and TNF alpha was detected in the rat cornea with method of Western blot protein detection at the same time.The differences of each group were analyzed,and the correlation was assessed between TLRs and inflammatory factor.Results There were the expressions of TLR-2,TLR-4,IL-1 beta and TNF alpha in the normal rat cornea,and its expressions were gradually increased after alkali burn,reached the peak at the 7 days,then decreased gradually,the difference of each group（the 3 day,the 7 day,the 14 day,the 21 day） was statistically significant （all P〈0.05）.The expressions of TLR-2 and IL-1 beta （r=0.986,P〈0.05）,TNF alpha （r=0.986,P〈0.05） were positive correlated.The expressions of TLR-4 and IL-1 beta（r=0.975,P〈0.05）,TNF alpha （r=0.990,P〈0.05） were positive correlated.Conclusion TLRs participates and may start immune inflammatory response after corneal alkali burn,mediates the production of inflammatory cytokine.