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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。
OBJECTIVE: To establish a model of iBRB in vitro and investigate the effect of retinal Müller glial cells (RMGC) on tight junction (TJ). Methods: Purified and cultured rat retinal microvascular endothelial cells (RMECs) were immunoprecipitated by immunomagnetic beads method. RMGC was cultivated by modified enzyme digestion method. The iBRB model was established by co-culture of RMEC and RMGC on both sides of microporous membrane. Transmission electron microscopy (TEM) was used to observe the growth of both cells and the morphology of TJ. Transient endothelial cell resistance (TER) measurement and TJ-related protein ZO-1 fluorescence staining and protein electrophoresis were used to evaluate the changes of TJ. Results: Both RMEC and RMGC could grow normally on the membrane with close junctions between RMGCs. TEM observation of high TER samples adjacent RMEC form a typical TJ structure. The TER of the RMEC layer reached the highest at 8 days (86.3 ± 7.9) Ω · cm 2, and remained relatively stable thereafter. (97.6 ± 3.6) Ω · cm2 in the presence of RMGC in the co-culture group and twice as high as that in the RMEC group at 7 days, reaching a peak value of (113.5 ± 5.3) Ω · cm2 at 8 days. During the development of iBRB model, the expression intensity of ZO-1 increased with time. CONCLUSION: In vitro iBRB model can be established by co-culture of RMEC and RMGC. RMGC can accelerate the TJ maturation of RMEC.