高糖对原代培养的视网膜神经节细胞中Toll样受体4表达的上调作用及其意义

Regulation of high glucose to the expression of Toll-like receptor 4 in retinal ganglion cells and its significance

背景研究发现糖尿病性视网膜神经病变的发生早于糖尿病视网膜病变（DR），视网膜神经节细胞（RGCs）的损伤是DR的早期特征性改变。研究表明，Toll样受体4（TLR4）在糖尿病大鼠视网膜中呈高表达，但TLR4与糖尿病性RGCs损伤的关系尚不明确。目的研究高糖对原代培养的大鼠RGCs中TLR4表达的影响，为糖尿病性视网膜神经病变的预防和治疗以及靶向药物研究提供依据。方法采用木瓜蛋白酶消化法从出生后1～3 d的SPF级大鼠视网膜中分离RGCs并进行纯化培养，采用免疫荧光技术检测RGCs特异性标志物Brn3a的表达以鉴定培养的细胞。将培养的细胞分为正常对照组及10、20、30 mmol/L葡萄糖组，分别于不同浓度葡萄糖培养后24 h及48 h收集细胞，分别采用实时荧光定量PCR法和Western blot法测定RGCs中TLR4 mRNA及其蛋白的相对表达量。结果细胞接种后贴壁生长并呈近圆形，纯化培养后24 h细胞体积逐渐增大且呈聚集样岛状生长，可见突触和轴突。培养的细胞中Brn3a为阳性表达。葡萄糖培养后24 h岛样细胞群周边细胞轮廓不清，反光弱，葡萄糖培养后48 h部分细胞内结构消失，仅残留细胞轮廓，可见大量细胞碎片。细胞培养后24 h，10、20和30 mmol/L葡萄糖组RGCs中TLR4 mRNA相对表达量分别为0.945±0.237、1.180±0.193和0.827±0.213，培养后48 h分别为1.509±0.422、2.433±0.617和1.435±0.410，均明显高于正常对照组的0.600±0.099和0.724±0.302，差异均有统计学意义（均P〈0.01）。细胞培养后24 h，10、20和30 mmol/L葡萄糖组RGCs中TLR4蛋白相对表达量分别为0.442±0.147、0.626±0.128和0.330±0.153，培养后48 h分别为0.464±0.121、0.930±0.441和0.394±0.158，均明显高于正常对照组的0.090±0.050和0.094±0.070，差异均有统计学意义（均P〈0.01）。结论高糖环境下大量体外培养的RGCs细胞内结构消失，同时细胞中TLR4表达量上调，提示RGCs中TLR4的过量表达可能与高糖诱导的RGCs损伤有关。

BackgroundStudies show that retinal neurodegeneration may precede retinal microvascular changes in diabetes mellitus.The apoptosis of retinal ganglion cells （RGCs） is an early finding in retinal neurodegeneration.Toll-like receptor 4 （TLR4） is proved to be up-regulated in diabetic rats retina.However, the impact of TLR4 on RGCs damage in retinal neurodegeneration is poorly understood.ObjectiveThe aim of this study was to investigate the expressing change of TLR4 induced by high glucose in RGCs in order to offer a basis for the prevention diabetic retinal neurodegeneration and the study on targeting drugs.MethodsRGCs were isolated and purified from the retinas of SPF SD rats aged postnatal 1-3 days by using papain digestion method and then were identified by immunofluorescence technology to detect the expression of Brn3a, a specific marker of RGCs.The cells were divided into normal control group and 10, 20, 30 mmol/L glucose groups.The expressions of TLR4 mRNA and protein in the cells were detected by real-time fluorescence quantitative PCR and Western blot analysis in 24 and 48 hours after addtion of glucose.All procedures performed in studies were in accordance with the Association for National Institutes of Health （NIH） Statement for the Care and Use of Laboratory Animals recommendations.The protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University.Every effort was made to minimize animal discomfort and stress.ResultsThe normal cells grew well with the shape of near roundness after inoculaton.The cells were gradually enlarged and clustered with obvious axons and dendrites 24 hours after purifying.Brn3a showed the positive expression in cultured cells.At 24 hours and 48 hours after glucose culture, the cell structures were gradually invisible in most cells.The expressions of TLR4 mRNA in the cells were 0.945±0.237, 1.180±0.193 and 0.827±0.213 at 24 hours and 1.509±0.422, 2.433±0.617 and 1.435±0.410 at 48 hours after culture in the 10, 20 and 30 mmol/L glucose groups, respectively, which were significantly higher than 0.600±0.099 and 0.724±0.302 in the normal control group （all at P〈0.01）. The expressions of TLR4 protein in the cells were 0.442±0.147, 0.626±0.128 and 0.330±0.153 at 24 hours and 0.464±0.121, 0.930±0.441 and 0.394±0.158 at 48 hours after culture in the 10, 20 and 30 mmol/L glucose groups, respectively, which were significantly higher than 0.090±0.050 and 0.094±0.070 in the normal control group （all at P〈0.01）.ConclusionsA large number of RGCs die in a high-glucose environment in vitro, meanwhile, the expression of TLR4 up-regulates in the cells, indicating that TLR4 maybe participate in the damage of RGCs induced by high glucose.