改良大鼠肺微血管内皮细胞原代培养技术及细胞鉴定

目的 改进大鼠肺微血管内皮细胞的原代培养方法,并对所培养的原代细胞进行鉴定.方法 应用SD大鼠的外周肺组织,进行大鼠肺微血管内皮细胞的原代培养.从动物选取、取材、肺灌注、植块贴壁、培养基及添加物的选择等方面对肺微血管内皮细胞原代培养技术方法进行改进.从细胞形态学特征,免疫组织化学检测血管内皮细胞表面标志物（CD31抗原和Ⅷ因子相关抗原的表达）,荧光显微镜观察异硫氰酸荧光标记植物凝集素与肺微血管内皮细胞特异性结合情况等方面鉴定所培养的肺微血管内皮细胞.流式细胞仪检测细胞纯度,噻唑蓝测量细胞生长曲线.结果 原代培养肺微血管内皮细胞成多角形,融合为单层后呈典型的鹅卵石或铺路石样生长,传代培养后可变为梭形呈漩涡样生长或聚集生长.免疫组织化学显示细胞CD31和Ⅷ因子相关抗原的表达阳性;异硫氰酸荧光标记植物凝集素结合试验阳性.细胞生长状态良好、细胞纯度达93.2％.结论 改进后的原代培养方法简便、可靠,获得的肺微血管内皮细胞污染少、纯度高,状态良好,保持了内皮细胞的结构和特性.

Improventment of method primary cultivation and identification of rat pulmonary microvascular endothelial cells

Objective To improve the method for primary cultivation of rat pulmonary microvascular endothelial cells （PMEVCs） and identify the primary cultivated cells.Methods PMVECs were derived from peripheral lung tissue of Sprague-Dawley rats.Primary cultivation method was improved from the procedures of animal selection,lung tissue perfusion,tissue piece pasting,culture medium selection and so on.Inverted microscope was used to observe morphological characteristics of pulmonary microvascular endothelial cells.Immunohistochemical staining for expression of Ⅷ-related antigen and CD31,and observed by fluorescence microscope for binding with lectin from BSI （FITC-BSI binding away） to identify pulmonary microvascular endothelial cells.The cell purity was detected with flow cytometry.Results The PMVECs were exhibited as polygon and presented typical cobblestone-like morphology after fusion to monolayer with contact inhibition.PMVECs turned to be fusiform and presented a swirling or aggregate growth pattern after transfer of culture.Immunohistochemical staining revealed that the expression of CD31 and factor Ⅷ-related antigen was positive.Besides,there were positive findings for FITC-BSI assay.The vitality and growth rate of PMVECs were in good condition.The cell purity was 93.2％ with the improvement method of primary cultivation.Condusions The improved methods for primary cultivation is easy to handle,with favorable repeatability and success rate.PMVECs obtained with this method will display lower contamination,higher purity,faster growth and better status.