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目的改进SD大鼠肺微血管内皮细胞(PMVECs)原代培养方法,以获得纯化的大鼠PMVECs。方法对组织块法培养PMVECs过程中的组织取材、植块贴壁、培养基配制、传代培养进行改进包括:在麻醉前提前注射肝素钠;利用腹腔放血,待大鼠停止呼吸后剪开胸腔;改进操作以及试剂中加入双抗以降低污染率;原代培养48h即取出组织块;进行再次消化去除成纤维细胞;异硫氰酸标记的植物凝集素(FITC-BSI)鉴定培养的PMVECs。培养后在倒置显微镜下观察细胞形态。结果原代培养的细胞呈现为多角形或短梭形,呈单层铺路石样排列的典型结构。随着细胞传代或培养条件的改变,细胞形态发生变化,可呈长梭形,漩涡状排列。FITC-BSI结合实验呈绿色荧光,阳性率达到90%以上。结论通过改进分离及培养方法可获得生长状态良好、纯度高且能够稳定传代培养的PMVECs。
Objective To improve primary culture of pulmonary microvascular endothelial cells (PMVECs) in SD rats to obtain purified rat PMVECs. Methods Tissue samples, explants adherent, medium preparation and subculturing in PMVECs during tissue block culture were improved including: injection of heparin sodium before anesthesia; use of intraperitoneal bloodletting to cut off the thorax after the rats stopped breathing; Improved operation and double anti-agent to reduce the contamination rate; primary cultured 48h remove the tissue block; re-digestion to remove fibroblasts; isothiocyanate labeled lectin (FITC-BSI) identification of cultured PMVECs. After culturing, the cell morphology was observed under an inverted microscope. Results The primary cultured cells showed polygonal or short fusiform shape with typical arrangement of monolayer paving stones. With the passage of cells or changes in culture conditions, changes in cell morphology can be long fusiform, swirling arrangement. FITC-BSI binding experiment was green fluorescence, the positive rate of more than 90%. Conclusion PMVECs with good growth status, high purity and stable subculture can be obtained by improving the isolation and culture methods.