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目的:用免疫磁珠法原代培养大鼠视网膜微血管内皮细胞(RMEC),观察共培养Müller细胞对视网膜微血管内皮细胞增生和迁移的影响。方法:采用免疫磁珠的细胞分选法分离大鼠视网膜微血管内皮细胞,在条件培养基中培养生长,采用第Ⅷ因子抗体免疫组化染色方法鉴定细胞,采用流式细胞术和台盼兰染色分析传代中的微血管内皮细胞纯度以及细胞活性。传统的方法培养并鉴定Müller细胞。采用不同孔径微孔滤膜培养小室,进行大鼠视网膜微血管内皮细胞与Müller细胞分离共培养,对照组培养环境中无Müller细胞。以细胞曲线描绘反映细胞增生,并用流式细胞仪测定细胞周期。相差显微镜下计算穿过微孔滤膜迁移贴附于背面的内皮细胞数量。结果:通过磁珠分离方法获得了纯度为97.13%大鼠视网膜微血管内皮细胞,细胞活力达92%,第Ⅷ因子抗体染色呈阳性。与Müller细胞共培养的RMEC可早于正常培养2～3d进入生长平台期。RMEC中S期和G2期比例增加,G1期比例相对减少。S、G2和G1期百分比,在共培养组24h分别为44.0%,11.2%和44.8%,48h分别为42.3%,10.9%和46.8%;对照培养组24h分别为41.3%,4.9%和53.8%,48h分别为40.1%,4.7%和55.2%。迁移的细胞数增加,共培养6h移行至膜下的内皮细胞数12.2±2.5个,12h为51.7±23.4个,对照培养6h移行至膜下的内皮细胞3.3±2.5个,12h为14.7±7.0个,6h和12h两组内皮细胞数差异具有统计学意义(P<0.05)。结论:免疫磁珠细胞分选方法可以获得高纯度的大鼠视网膜微血管内皮细胞,且对细胞活力无影响。Müller细胞可以促进视网膜内皮细胞的迁移,促进该细胞的增生。
OBJECTIVE: To primarily culture rat retinal microvascular endothelial cells (RMECs) by immunomagnetic beads method and observe the effect of co-cultured Müller cells on the proliferation and migration of retinal microvascular endothelial cells. METHODS: Rat retinal microvascular endothelial cells were isolated by cell sorting using immunomagnetic beads and cultured in conditioned medium. The cells were identified by immunohistochemical staining with factor Ⅷ antibody. Flow cytometry and Trypan blue staining The purity and cell viability of microvascular endothelial cells during passage were analyzed. Traditional methods to culture and identify Müller cells. Using different pore size microporous membrane culture chamber, the rat retinal capillary endothelial cells and Müller cells were isolated and co-cultured, the control group cultured without Müller cells. The cell curve is plotted to reflect cell proliferation, and the cell cycle is measured by flow cytometry. The number of endothelial cells attached to the back surface through the microporous membrane migration was calculated by phase contrast microscopy. Results: The retinal capillary endothelial cells with a purity of 97.13% were obtained by magnetic bead separation method. The cell viability reached 92%, and the factor Ⅷ antibody staining was positive. RMEC co-cultured with Müller cells can enter the growth plateau earlier than normal culture for 2-3 days. The proportion of S phase and G2 phase increased in RMEC, while the proportion in G1 phase decreased relatively. The percentages of S, G2 and G1 were 44.0%, 11.2% and 44.8% respectively in the co-culture group and 42.3%, 10.9% and 46.8% in 48h and 41.3%, 4.9% and 53.8% , 48h respectively 40.1%, 4.7% and 55.2%. The number of migrating cells increased, the number of endothelial cells migrating to sub-membrane at 6h after co-culture was 12.2 ± 2.5, and the number of endothelial cells at 12h was 51.7 ± 23.4, and the number of endothelial cells under sub-membrane migration was 3.3 ± 2.5 at 14h and 14.7 ± 7.0 6h and 12h endothelial cell number difference was statistically significant (P <0.05). CONCLUSION: The immunomagnetic bead cell sorting method can obtain high-purity rat retinal microvascular endothelial cells and has no effect on cell viability. Müller cells can promote the migration of retinal endothelial cells and promote the proliferation of the cells.