中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
11期
1045-1049
,共5页
Toll样受体4%视神经/损伤%炎症%视网膜/神经节细胞
Toll樣受體4%視神經/損傷%炎癥%視網膜/神經節細胞
Toll양수체4%시신경/손상%염증%시망막/신경절세포
Toll-like receptor 4%Optic nerve/crush%Inflammation%Retina/ ganglion cell
背景 Toll样受体4(TLR4)是一种重要的免疫相关受体,在多种疾病的发生中起致炎作用.研究发现,视神经损伤后继发的炎症反应可进一步引起视网膜损伤,因此视神经损伤后TLR4的表达及其效应值得研究. 目的 研究大鼠视神经损伤后视网膜TLR4的表达情况. 方法 选取成年健康SPF级SD大鼠24只,按随机数字表法随机分为视神经损伤3d组和视神经损伤7d组.取大鼠右眼用视神经钳夹法制备视神经损伤模型,左眼不予处理为对照组.分别于视神经损伤后3d和7d用过量麻醉法处死大鼠并分离视网膜,采用免疫荧光法检测各组大鼠视网膜中TLR4的表达；分别采用逆转录PCR法(RT-PCR)和Western blot法检测大鼠视网膜中TLR4 mRNA及其蛋白的表达；采用TUNEL染色法观察各组大鼠视网膜神经节细胞(RGCs)的凋亡情况.结果 视网膜免疫荧光法检测结果显示,TLR4在大鼠视网膜中呈绿色荧光,视神经损伤3d组和视神经损伤7d组造模眼视网膜中的荧光强度较对照组左眼均明显增强,绿色荧光主要分布在视网膜内层.RT-PCR法检测表明,模型眼视网膜损伤后3d和7d视网膜中TLR4 mRNA相对表达量分别为2.92±0.06和3.92±0.12,对照眼TLR4 mRNA的相对表达量分别为2.87±0.12和3.44±0.17,大鼠模型眼TLR4 mRNA表达的灰度值较对照眼明显增加,差异均有统计学意义(t3d=-12.888,P＜0.001；t7d=-4.669,P=0.010).Western blot法检测显示,大鼠模型眼视网膜损伤3d和7d视网膜中TLR4蛋白的相对表达量分别为1.14±0.05和1.49±0.03,对照眼TLR4蛋白的相对表达量分别为0.99±0.09和1.38±0.07,模型眼视网膜中TLR4蛋白表达量明显高于对照眼,差异均有统计学意义(t3d=-11.324,P＜0.001；t7d=-5.638,P=0.005).TUNEL染色显示,模型眼RGCs凋亡数较对照眼增多. 结论 TLR4在视神经损伤大鼠视网膜内层的表达明显上调,提示TLR4通路可能参与RGCs的损伤.
揹景 Toll樣受體4(TLR4)是一種重要的免疫相關受體,在多種疾病的髮生中起緻炎作用.研究髮現,視神經損傷後繼髮的炎癥反應可進一步引起視網膜損傷,因此視神經損傷後TLR4的錶達及其效應值得研究. 目的 研究大鼠視神經損傷後視網膜TLR4的錶達情況. 方法 選取成年健康SPF級SD大鼠24隻,按隨機數字錶法隨機分為視神經損傷3d組和視神經損傷7d組.取大鼠右眼用視神經鉗夾法製備視神經損傷模型,左眼不予處理為對照組.分彆于視神經損傷後3d和7d用過量痳醉法處死大鼠併分離視網膜,採用免疫熒光法檢測各組大鼠視網膜中TLR4的錶達；分彆採用逆轉錄PCR法(RT-PCR)和Western blot法檢測大鼠視網膜中TLR4 mRNA及其蛋白的錶達；採用TUNEL染色法觀察各組大鼠視網膜神經節細胞(RGCs)的凋亡情況.結果 視網膜免疫熒光法檢測結果顯示,TLR4在大鼠視網膜中呈綠色熒光,視神經損傷3d組和視神經損傷7d組造模眼視網膜中的熒光彊度較對照組左眼均明顯增彊,綠色熒光主要分佈在視網膜內層.RT-PCR法檢測錶明,模型眼視網膜損傷後3d和7d視網膜中TLR4 mRNA相對錶達量分彆為2.92±0.06和3.92±0.12,對照眼TLR4 mRNA的相對錶達量分彆為2.87±0.12和3.44±0.17,大鼠模型眼TLR4 mRNA錶達的灰度值較對照眼明顯增加,差異均有統計學意義(t3d=-12.888,P＜0.001；t7d=-4.669,P=0.010).Western blot法檢測顯示,大鼠模型眼視網膜損傷3d和7d視網膜中TLR4蛋白的相對錶達量分彆為1.14±0.05和1.49±0.03,對照眼TLR4蛋白的相對錶達量分彆為0.99±0.09和1.38±0.07,模型眼視網膜中TLR4蛋白錶達量明顯高于對照眼,差異均有統計學意義(t3d=-11.324,P＜0.001；t7d=-5.638,P=0.005).TUNEL染色顯示,模型眼RGCs凋亡數較對照眼增多. 結論 TLR4在視神經損傷大鼠視網膜內層的錶達明顯上調,提示TLR4通路可能參與RGCs的損傷.
배경 Toll양수체4(TLR4)시일충중요적면역상관수체,재다충질병적발생중기치염작용.연구발현,시신경손상후계발적염증반응가진일보인기시망막손상,인차시신경손상후TLR4적표체급기효응치득연구. 목적 연구대서시신경손상후시망막TLR4적표체정황. 방법 선취성년건강SPF급SD대서24지,안수궤수자표법수궤분위시신경손상3d조화시신경손상7d조.취대서우안용시신경겸협법제비시신경손상모형,좌안불여처리위대조조.분별우시신경손상후3d화7d용과량마취법처사대서병분리시망막,채용면역형광법검측각조대서시망막중TLR4적표체；분별채용역전록PCR법(RT-PCR)화Western blot법검측대서시망막중TLR4 mRNA급기단백적표체；채용TUNEL염색법관찰각조대서시망막신경절세포(RGCs)적조망정황.결과 시망막면역형광법검측결과현시,TLR4재대서시망막중정록색형광,시신경손상3d조화시신경손상7d조조모안시망막중적형광강도교대조조좌안균명현증강,록색형광주요분포재시망막내층.RT-PCR법검측표명,모형안시망막손상후3d화7d시망막중TLR4 mRNA상대표체량분별위2.92±0.06화3.92±0.12,대조안TLR4 mRNA적상대표체량분별위2.87±0.12화3.44±0.17,대서모형안TLR4 mRNA표체적회도치교대조안명현증가,차이균유통계학의의(t3d=-12.888,P＜0.001；t7d=-4.669,P=0.010).Western blot법검측현시,대서모형안시망막손상3d화7d시망막중TLR4단백적상대표체량분별위1.14±0.05화1.49±0.03,대조안TLR4단백적상대표체량분별위0.99±0.09화1.38±0.07,모형안시망막중TLR4단백표체량명현고우대조안,차이균유통계학의의(t3d=-11.324,P＜0.001；t7d=-5.638,P=0.005).TUNEL염색현시,모형안RGCs조망수교대조안증다. 결론 TLR4재시신경손상대서시망막내층적표체명현상조,제시TLR4통로가능삼여RGCs적손상.
Background Toll-like receptor 4 (TLR4) is an immune related receptor.It plays an important role in inducing inflammation response.The inflammatory response secondary to optic nerve crush will results in serious retinal damage.It is worthy of studying the expression and effect of TLR4 in retina after optic nerve crush.Objective This experimental study was to explore the role of TLR4 in the loss of retinal ganglion cells(RGCs) after optic nerve crush.Methods Twenty-four SPF adult health Sprague-Dawley (SD) rats were used in the study and radomized into two groups based to the experimental time.The optical nerve crush models were established by crushing the optical nerve in the right eyes of the rats,and the left normal eyes served as controls.The rats were sacrificed by over anesthesia and retinas were isolated 3 days and 7 days after operation.Expression of TLR4 in the retinas was detected using immunofluorescence method.Reverse trancription PCR (RT-PCR) and Western blot were applied respectively for the detection of TLR4 mRNA and protein in the retinas.The apoptosis of RGCs was evaluated using TUNEL staining.The use and care of experimental animals followed the " Guide for the Care and Use of Laboratory Animals" of NIH.This study was approved by the Institutional Animal Care and Use Committee at the Zhongshan Ophthalmic Center.Results The expression of TLR4 in rat retinas presented with green fluorescence mainly in the inner layer of retinas.The fluorescence was enhanced in the model 3 days group and the model 7 days group compared with the corresponding control groups.The relative expressing values of TLR4 mRNA in the retinas were 2.92±0.06and 3.92±0.12 in the model 3 days and 7 days groups,respectively,which were significantly higher than 2.87±0.12and 3.44±0.17 in the control 3 days and 7 days group (t3d =-12.888,P＜0.001 ;t7d =-4.669,P=0.010).In the model 3 days group and model 7 days group,the grey values of TLR4 protein were 1.14±0.05 and 1.49±0.03,and those in the control 3 days and 7 days groups were 0.99 ± 0.09 and 1.38 ± 0.07,showing significant differences between them(t3d =-11.324,P＜0.001 ;t7d =-5.638,P=0.005).Apoptotic RGCs were obviously increased in the optic nerve damage group in comparison with the control group.Conclusions The TLR4 is over-expressed in the inner layer of retina after optic nerve crush,which suggestes that TLR4 is probably involved in the loss of RGCs after optic nerve crush.
