体外内层血视网膜屏障模型中Müller胶质细胞对紧密连接的影响

Effects of retinal Müller glial cells on the tight junctions in an in vitro model of the inner blood retinal barrier

目的:建立体外内层血视网膜屏障(iBRB)模型并研究视网膜Müller胶质细胞(RMGC)对紧密连接(TJ)的影响。方法:采用免疫磁珠方法原代纯化和培养大鼠视网膜微血管内皮细胞(RMEC),采用改良酶消化方法培养RMGC,RMEC和RMGC在微孔膜的两侧共培养建立iBRB模型,通过相差显微镜和透射电子显微镜(TEM)观察两种细胞的生长和TJ的形态。采用跨内皮细胞电阻(TER)测量和TJ相关蛋白ZO-1荧光染色和蛋白电泳来评价TJ的变化情况。结果:RMEC和RMGC均可在膜上正常生长,RMGC间紧密连接。TEM观察高TER值样品中相邻RMEC形成典型的TJ结构。RMEC层TER在8d时达到最高,为(86.3±7.9)Ω·cm2,此后保持相对稳定。在RMGC存在的共培养组TER7d时为(97.6±3.6)Ω·cm2,约为7d时RMEC组的2倍,8d达到峰值(113.5±5.3)Ω·cm2。在iBRB模型发展过程中,ZO-1的表达强度随时间延长而增加。结论:通过RMEC和RMGC的共培养可以建立体外iBRB模型,RMGC加快RMEC间的TJ成熟。

AIM： To establish an in vitro model of the inner blood retinal barrier （iBRB） and to observe the effects of retinal Huller glial cells （RMGC） on the tight junctions in the model. METHODS： This in vitro BRB model is composed by the retinal microvascular endothelial cells （RMEC） and RMGC of rats. RMEC were isolated with magnetic cell sorting （MACS） system and cultured in conditioned medium with endothelial cell growth supplements. RMGC were cultivated and purified by modified digest method. RMEC and RMGC were seeded on different surfaces of special insert filters and co-cultured in RMEC medium. Morphologies of the two cells＇ growth were studied with phase contrast microscopy and tight junction was observed with transmission electron microscopy （TEM）. Tight junction （TJ） was evaluated by transendothelial electrical resistance （TER） measurements and TJ associated protein, zonula occludens protein 1 （ZO-1） which was detected by immunofluorescence staining and Western blot. RESULTS： Both RMEC and RMGC grew on surfaces and formed mature tight junctions. TEM pictures of the high-resistance cultures revealed that neighboring cells were closely apposed with one another with the formation of typical tight junctions. TER of RNEC monolayer reached （86.3±7.9）Ω·cm^2 on day 8 and remained fairly constant. But in co-culture group, TER of RNEC layer with RNGC reached （97.6±3.6）Ω·cm^2 on day 7, which was about doubled that of control EC monolayer and remained constant, peaking at （113.5± 5.3） Ω·cm^2 in the following days. During the development of BRB model, there was time-dependent intensity of expression of ZO-1. CONCLUSION： The in vitro model of the inner blood retinal barrier has been established successfully. RMGC enhance evolution of tight junctions between RMEC.