Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07
Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07
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DOI：
10.1167/iovs.07-1522
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Xu, Heping
中科院分区：
文献类型：
--
作者：
Chen, Mei;Muckersie, Elizabeth;Xu, Heping
目的。从小鼠视网膜色素上皮细胞原代培养中克隆出一株自发生成的视网膜色素上皮（RPE）细胞系（B6-RPE07），并在培养中维持了18个月以上。对该细胞系的形态学和功能特性进行了鉴定。采用相差光镜、电子显微镜、共聚焦显微镜观察B6-RPE07细胞形态。通过从培养室的顶端到基底外侧的荧光通量来测量屏障性能。通过共聚焦显微镜、电镜和流式细胞术检测细胞结合/吞噬光感受器外段（POS）的能力。细胞因子/趋化因子分泌量采用细胞计数头阵列检测。RT-PCR和Western blot检测视觉周期蛋白的表达。在标准培养条件下，B6-RPE07细胞呈鹅卵石状形态。当在三维（3D）胶原凝胶膜上培养时，B6-RPE07细胞表现出单层上皮极化，顶端表面有微绒毛。B6-RPE07培养物免疫组化显示泛细胞角蛋白高表达。B6-RPE07细胞也表达视网膜色素上皮特异性标志物CRALBP，但不表达RPE65。B6-RPE07细胞中检测到细胞连接蛋白ZO-1和β -catenin，但未检测到claudin-1/3和occludin-1。B6-RPE07细胞能够结合、吞噬和消化POS，最终，B6-RPE07细胞产生高水平的IL-6和ccl2。这是首次报道的小鼠RPE细胞系，其形态，表型和功能与体内小鼠RPE细胞相似。该细胞系将成为未来RPE研究的宝贵资源，特别是体内基因修饰和移植研究。
PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel-coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and beta-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.