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用胶原酶消化,梯度高心,LDL-FACS法分离及纯化小鼠肺组织微血管内皮细胞(MLMEC),并根据细胞形态及间接免疫荧光法加以鉴定。纯化后的MLMEC形态为多边形、短梭形,呈鹅卵石样排列;它能摄取RB-DiL-Ac-LDL,从而胞浆内出现亮红色颗粒;用抗PECAM抗体间接免疫荧光染色后细胞接触处呈亮绿色,细胞轮廓清晰可见。用感染法将HSV-TK基因转入MLMEC细胞内,用XTT法显示携带有HSV-TK基因的MLMEC/TK细胞对GCV敏感,引起自杀效应。该分离、纯化及基因转移方法的建立将为研究MLMEC在各种病理状况下的作用及作为基因治疗的载体提供体外模型。
Mouse lung tissue microvascular endothelial cells (MLMECs) were isolated and purified by collagenase digestion, high-gradient centrifugation and LDL-FACS, and identified by cell morphology and indirect immunofluorescence. The purified MLMECs were polygonal, short fusiform and arranged in a cobblestone-like pattern. RB-DiL-Ac-LDL could be taken up to produce bright red granules in the cytoplasm. After indirect immunofluorescence staining with anti-PECAM antibody, Bright green, the cell outline clearly visible. The HSV-TK gene was transfected into MLMEC cells by infection method. The results showed that MLMEC / TK cells carrying HSV-TK gene were sensitive to GCV by XTT method, causing suicide effect. This separation, purification and establishment of gene transfer methods will provide an in vitro model for studying the role of MLMEC in various pathological conditions and as a vector for gene therapy.