大鼠肺微血管内皮细胞原代培养方法的改进及细胞鉴定

目的改进大鼠肺微血管内皮细胞（PMVECs）的原代培养方法,并对培养细胞进行相关鉴定。方法从动物选择、取材、循环灌注、植块贴壁等方面对体外分离PMVECs的原代培养方法进行改良。原代培养细胞经传代、冻存与复苏,倒置显微镜观察培养细胞的形态学特征和生长特性,免疫组织化学染色检测血管内皮细胞表面标志物Ⅷ因子相关抗原和CD31表达,荧光显微镜观察细胞与PMVECs的特异性结合物植物凝集素BSI的结合情况（FITC-BSI结合试验）。结果倒置显微镜下,培养细胞呈多边形或梭形,融合为单层后呈典型的鹅卵石或铺路石样镶嵌生长并有接触抑制现象;免疫组织化学染色显示,细胞CD31和Ⅷ因子表达阳性;FITC-BSI结合试验呈阳性结果。结论改进的原代细胞培养方法操作简单、可重复性好且成功率高;综合鉴定表明培养细胞形态学及生物学特性与PMVECs一致。

Improvement of method for primary cultivation and identification of rat pulmonary microvascular endothelial cells

Objective To improve the method for primary cultivation of rat pulmonary microvascular endothelial cells (PMVECs), and identify the cultivated cells. Methods Method for primary cultivation of rat PMVECs was improved from the procedures of animal selection, sampling, perfusion and tissue piece pasting. After subculturing, freezing and thawing, PMVECs were observed by inverted microscopy for morphology and growth behavior, identified by immunohistochemical staining for expression of factor Ⅷ and CD31, and observed by fluorescence microscopy for binding with lectin from BSI (FITC-BSI binding assay). Results Under inverted microscope, the cultivated cells exhibited as polygon or fusiform, and displayed typical cobblestone-like morphology after fusion to monolayer, with contact inhibition. Immunohistochemical staining revealed that the expression of factor Ⅷ and CD31 was positive. Besides, there were positive findings for FITC-BSI assay. Conclusion The improved method for primary cultivation is easy to handle, with favourable repeatability and success rate, and the morphological and biological characteristics of cultivated cells are in line with PMVECs.