微血管内皮细胞的分离、纯化、鉴定及基因转移的研究

A STUDY ON THE SEPARATION,PURIFICATION AND IDENTIFICATION OF MOUSE MICRO-VASCULAR ENDOTHELIAL CELL AND THE GENE TRANSFER

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 micro-vascular endothelial cell (MLMEC) was isolated and purified with the methods of collagenase digestion and LDL-FACS. The characterization of MLMEC was confirmed base on the morphology and indirect im-munofluorescence technology. Morphologically, MLMEC was polygonal, cuboidal, with cobblestone pattern,all cells took up RB-DiL-Ac-LDL that imparted brightly red granular fluorescent in their cytoplasm. For indirect immunofluo-rescence technology, the MLMEC bound the PECAM antibody to show the green fluorescent .which highlights their intercellular contacts to make the cell contour clear. With the method of infection,the HSV-TK gene was transferred to MLMEC. The sensitivity of TK gene transferred cell to GCV has been tested with XTT. The methods allowed the production of pure MLMEC and gransfer in this study that may prove a useful model in -vitro for study of the role of MLMEC in pathologic states and as a vehicle for gene therapy.