牛视网膜微血管周细胞和内皮细胞的选择性培养

Selective culture of bovine retinal capillary endothelial cells and pericytes

目的 建立视网膜微血管周细胞 (PC)和内皮细胞 (EC)选择性培养的简便方法。方法 采用含 2 0 %胎牛血清 (FBS)的DMEM和在该培养基中加入 5 %贫血小板人血浆 (PPP)及牛视网膜浸出液 (2 0 μl/ml) ,结合原代培养细胞克隆的分离、除杂。结果 获得的PC和EC能连续传代 ,纯净度 >95 %。PC形状不规则 ,无接触性抑制 ,对 3G5和α 肌动蛋白单克隆抗体染色阳性 ,Ⅷ因子抗体染色阴性 ;EC呈鹅卵石状生长 ,有接触性抑制 ,对Ⅷ因子抗体染色阳性 ,3G5和α 肌动蛋白抗体染色阴性。结论 用 2 0 %FBS的DMEM或在该培养液中加入 5 %PPP及视网膜浸出液 ,结合原代细胞克隆的分离、除杂 。

ObjectiveTo establish a simply and conveniently selective cultural method for bovine retinal endothelial cells and pericytes.Methodsmedia of DMEM contained 20% fetal bovine serum(FBS)or added 5% human platelet poor plasma(PPP)and 20 μl/ml retinal extract were used,which were separately combined with separating and weeding for the colonies of cells in primary culture.ResultsRelative pure retinal capillary pericytes and endothelial cells(purity>95%)obtained separately could be passaged serially.The pericytes were characterized by highly irregular peripheries,and non contact inhibited,overlapping pattern of growth at confluence,and positive staining for a monoclonal antibody 3G5 and α isoform of smooth muscle actin,but negative staining to factor Ⅷ antibody.The endothelial cells grew as contiguous isoland of cells,formed a contact inhibited monolayer at confluence,stained by antisera to factor Ⅷ antibody,and negative stained to antibody 3G5 and α isoform of smooth muscle actin.ConclusionIt can obtain purified endothelial cells and pericytes cultures using media of DMEM contained 20%FBS or adding 5%PPP and 20 μl/ml retinal extract in the media respectively combined with colon separating and weeding of cell in the primary culture