大鼠视网膜微血管内皮细胞的分离和培养

Isolation and culture of rat retinal capillary endothelial cells in vitro

目的探讨大鼠视网膜毛细血管内皮细胞(rat retinal capillary endothelial cells,RRCEC)的体外分离和选择性培养方法。方法采用单纯视网膜剪碎法获得的视网膜微血管碎片,结合0.25%胶原酶消化,经细胞筛网过滤,原代细胞悬液接种于纤维粘连蛋白(fibronectin,FN)包被的培养皿中,培养液为含20%胎牛血清(FBS)、100×103U·L-1肝素钠、10mg·L-1碱性成纤维细胞生长因子(bFGF)的DMEM液。原代培养5～6d细胞较少时,采用机械除杂法进行细胞分离,而在传代时利用进行选择性消化和贴壁法除杂分离以获得较纯RRCEC。应用Ⅷ因子相关抗体通过免疫组化方法对所培养的RRCEC进行鉴定。结果大鼠视网膜组织经过胶原酶适当消化后,产生大量具有良好生长能力的微血管碎片,贴壁后可见RRCEC自微血管碎片中游出,融合后呈铺路石样单层生长。传代培养时5～6d可融合生长。选择性培养获得的RRCEC纯度高,能连续传代,保持性状不变。对Ⅷ因子相关抗体染色阳性。结论视网膜剪碎、胶原酶、FBS、bFGF、培养皿处理及机械除杂法分离,选择性消化和贴壁等应用可获得较纯的RRCEC,方便简单,具有良好的重复性。

Objective To establish a simple method for isolation and cul- ture of rat retinal capillary endothelial cells（RRCEC） in vitro.Methods Fresh SD rat eyes were bisected posterior to the limbus and vitreous was removed by gentle traction with forceps. Retinas were detached from the optic nerve and cut into small segments.After applying 0,25% collagenase digestion, the suspension was passed through the double filtration with 100 μm nylon mesh.The microvessel suspension was cultured in a fibronectin （FN） coated substrate with DMEM supplemented with 20% fetal bovine serum （FBS）, 10 mg· L^-1 bFGF, 100 × 10^3 U· L^-1 heparin. At 5 -6 days, because the primary cultured cells were not too much, so mechanical cell weeding and seperation can be used. When passaging, as the character of RRCEC, selective digestion and attachment can be utilized to pure endothelial cells.Immunocytochemtstry was performed using a monoclonal antibody against the Ⅷ factor for RRCEC identification. Results After digestion, a lot of single small microvesse fragments could be seen under the phase contrast microscope.When these fragments attached onto the wells, the RRCEC travelled from them.The cells in primary culture began to clone in 3--7 days and then grew to confluence with 2 weeks and demonstrated a cobblestone morphology, focally forming contact-inhibited monolayers. But the passaged cells confluence only 6 days. The purity of selectively culture RRCEC was high, being reproducible,and positive staining for factor W antibody.Conclusion RRCEC with high purity can be obtained by using collagenase digestion, FBS, bFGF, selective cell weeding and coated -dished which are simple and repeatable methods. [ Rec Adv Ophthalmol 2005; 25 （5） ： 396-399 ]