人脐带内皮祖细胞的分离、鉴定及转染EGFP质粒的实验研究

Isolation, identification and EGFP gene transfection of endothelial progenitor cells derived from human umbilical cord

目的从脐带中分离内皮祖细胞（EPCs），考察其体外增殖、基因转染绿色荧光蛋白质粒的行为。方法以酶消化法从脐带中分离出内皮祖细胞，并通过流式细胞仪和共聚焦显微镜对内皮祖细胞进行鉴定，以Lipofectamine2000为转染试剂考察了内皮祖细胞转染绿色荧光蛋白质粒的行为。结果从脐带中分离培养的内皮祖细胞在第9天形成了典型的内皮细胞集落，流式细胞仪分析结果显示CD133和激酶插入区受体（KDR）的含量均有所提高，并具有内皮祖细胞结合异硫氰酸荧光素（FITC）标记的荆豆凝聚素1（FITC—UEA-1）和吞噬DiI标记的低密度脂蛋白（DiI—ac—LDL）的功能，能较好地表达绿色荧光蛋白。结论从脐带中分离的内皮祖细胞体外在适当的培养条件下，可增殖、诱导分化为内皮细胞，并能较好地表达外源基因，是基因与细胞治疗理想的载体。

Objective To isolate and identify endothelial progenitor cells （EPCs） from human umbilical cord, and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro. Methods EPCs were isolated from human umbilical cord in enzyme digestion method. The biological characteristics of EPCs were identified by flow cytometry and laser eonfoeal microscope. The enhanced green fluorescent protein （EGFP） gene transfection mediated by EPCs was investigated using Lipofeetamine 2000 as transfeetion reagent. Results Endothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later. These cells displayed an improved positive expression of CD133 and kinase insert domain receptor （KDR）. The endothelial- lineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate （FITC）-UEA-1 binding and DiI- ac-LDL uptake assay with the aid of laser confoeal microscope. The transfection results demonstrated high expression of EGFP taking EPCs as host cell. Conclusion Endothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions. EPCs demonstrated to be an ideal carrier for gene and cell therapy.