CAJ | 학술논문

背景 TLR-4是一种天然免疫受体,在免疫性中枢神经系统损伤的修复中发挥重要作用,但在青光眼视神经损伤中的具体作用机制不详.目的从基因和蛋白质水平研究正常大鼠及慢性高眼压模型大鼠视网膜组织中TLR-4的表达情况,探讨高眼压视网膜神经节细胞(RGCs)损伤的免疫机制及TLR-4在RGCs修复中的可能作用.方法选择4-5周龄的雄性清洁级纯种SD大鼠150只,用烧烙3条巩膜静脉联合术中应用丝裂霉素的方法建立大鼠慢性高眼压模型.Tono-penⅡ型笔式眼压计测量造模前、造模后2 h,1、3、7、14、28、56 d大鼠眼压.上述各时间点分别取5只高眼压组及空白对照组大鼠视网膜行免疫组织化学染色,观察视网膜TLR-4蛋白的表达情况.应用逆转录聚合酶链反应(RT-PCR)法及Western blot法检测各组视网膜组织中TLR-4 mRNA及蛋白质的表达.结果实验组手术后眼压明显升高,与空白对照组比较差异有统计学意义(P<0.01).免疫组织化学检测结果显示造模后早期TLR-4在视网膜组织中的表达开始增加,表达量均高于空白对照组(P<0.05-0.01);RT-PCR检测表明,大鼠慢性高眼压模型眼造模后各时间点TLR-4 mRNA在视网膜的表达量明显高于空白对照组(P<0.05-0.01);Western blot检测显示TLR-4在造模后早期视网膜组织中即有表达,结果显示组间差异有统计学意义(P<0.05-0.01).结论 TLR-4在大鼠慢性高眼压模型眼视网膜中的表达明显上调,提示其在大鼠慢性高眼压视神经退行性病变中发挥免疫调节作用.
배경 TLR-4시일충천연면역수체,재면역성중추신경계통손상적수복중발휘중요작용,단재청광안시신경손상중적구체작용궤제불상.목적 종기인화단백질수평연구정상대서급만성고안압모형대서시망막조직중TLR-4적표체정황,탐토고안압시망막신경절세포(RGCs)손상적면역궤제급TLR-4재RGCs수복중적가능작용.방법선택4-5주령적웅성청길급순충SD대서150지,용소락3조공막정맥연합술중응용사렬매소적방법건립대서만성고안압모형.Tono-penⅡ형필식안압계측량조모전、조모후2 h,1、3、7、14、28、56 d대서안압.상술각시간점분별취5지고안압조급공백대조조대서시망막행면역조직화학염색,관찰시망막TLR-4단백적표체정황.응용역전록취합매련반응(RT-PCR)법급Western blot법검측각조시망막조직중TLR-4 mRNA급단백질적표체.결과실험조수술후안압명현승고,여공백대조조비교차이유통계학의의(P<0.01).면역조직화학검측결과현시조모후조기TLR-4재시망막조직중적표체개시증가,표체량균고우공백대조조(P<0.05-0.01);RT-PCR검측표명,대서만성고안압모형안조모후각시간점TLR-4 mRNA재시망막적표체량명현고우공백대조조(P<0.05-0.01);Western blot검측현시TLR-4재조모후조기시망막조직중즉유표체,결과현시조간차이유통계학의의(P<0.05-0.01).결론 TLR-4재대서만성고안압모형안시망막중적표체명현상조,제시기재대서만성고안압시신경퇴행성병변중발휘면역조절작용.
Background TLR-4 is a natural immunity receptors in immunity,and it plays an important role in the repair of central nervous system damage.But its effect in glaucoma optic nerve injury is unclear.Objective This study was to investigate the expression of TLR-4 in retina with high intraocular pressure(IOP)in genetic and Drotein level and therefore explore the mechanism of TLR-4 on retinal ganglion cells(RGCs)injury. Methods Chronic ocular hypertension models were established in the right eyes of 150 clean purebred Sprague-Dawley rats by cauterizing the 3 sallow sclera veins.IOP was measured before and after 2 h,1 day,3,7,14,28,56 days after operation by PEN Ⅱ TONO-type pen tonometer.The expression of TLR-4 protein in rat retina was detected by immunohistochemistry and Western blot,and expression of TLR-4 mRNA was assayed by real time-PCR.This experimental procedure foliowed the Statement of Association for Research in Vision and Ophthalmology. Results The IOP was elevated in various time points after operation in experimental group,showing significant differences in comparison with control group(P<0.01).The immunohistochemistry revealed that the expression of TLR-4 protein in rat retina with chronic hypertension in 2 h,1 day,3,7,14,28,56 days after operation with the high A298 values in comparison with control eyes(P<0.05-0.01).Increased levels of TLR-4 mRNA in rat retinas were detected by RTPCR in high IOP eyes compared with control eyes in all time points after operation,presenting statistically significant differences between two groups(P<0.05-0.01).Western blot detection displayed the high expression of TLR-4 in retina in high IOP eyes early after operation with statistically significant results between model group and control group (P<0.05-0. 01). Conclusion TLR-4 is up-regulated in rat retina with chronic high IOP,suggesting that TLR-4 plays an immunoregulatory effect in glaucomatous eye.