流式细胞术检测人／山羊嵌合体中人源细胞方法的建立与应用  

Establishment and application of flow cytometry in detecting human cells in human/goat chimera models

作　　者：王娟[1] 陈美珏[1] 巩芷娟[1] 任兆瑞[1] 曾凡一[2] 黄淑帧[1] 

机构地区：[1]上海交通大学医学院附属上海市儿童医院上海医学遗传研究所,200040 [2]上海交通大学医学院医学科学研究院

出　　处：《中华检验医学杂志》2009年第4期441-445,共5页Chinese Journal of Laboratory Medicine

基　　金：国家重大科学研究计划资助项目（2007CB947800）;国家及上海市重点学科资助项目（B204）.志谢 美国Becton Dickinson公司的张珏、王练工程师和深圳市输血医学研究所熊文医师在流式细胞仪分选和分析方面给予了帮助,上海交通大学附属第六人民医院产科和上海市脐带血造血干细胞库在提供脐血方面给予了支持.

摘　　要：目的建立应用流式细胞术检测人／山羊嵌合体中人源细胞的方法。方法通过宫内移植的方法将标记有绿色荧光蛋白（GFP）的人造血干／祖细胞（MIG细胞）移植入胎山羊腹腔内，从而获得人／山羊嵌合体模型（MIG羊）。采集MIG羊的外周血，以数十种鼠抗人的抗体标记，筛选出对人源细胞特异性强而与山羊细胞无或仅有微弱交叉反应的抗体，列人流式细胞仪常规检测的首选抗体，继而在非移植山羊的外周血中加入不同比例（25％、50％、75％和100％）的人脐血细胞，用CD34^＋表面抗体标记来观察人脐血细胞和人CD34细胞的分布，以决定“门”的区域和大小；取MIG羊的肝脏，应用流式细胞术检测灌流后肝实质细胞中GFP细胞的比例及DNA含量。结果筛选出CD7、CD15、CD38、CD45、CD20、CD34、CD14和血型糖蛋白A（GPA）等8种单抗作为检测嵌合体中人源细胞的首选抗体；确定了进行流式细胞分析时“门”的大小和区域；MIG羊肝实质细胞中，GFP^＋细胞的比例为29．1％（29100／100000）；DNA含量测定结果显示MIG羊肝组织分选获得的GFP^＋细胞主要呈现2个峰，其位置与人的双峰位置相对应。结论流式细胞术是研究干细胞在体内分化、归巢和生物学特征的快速、简便、有效的方法；表面抗体以及“门”的选择对于准确检测人／山羊嵌合体中人源细胞至关重要。Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerism. Methods Human hemopoietic stem/progenitor cells （CD34^＋ ceils ） or MIG- transduced-GFP CD34^＋ cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera model. The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoclonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry. Human cord blood was proportionally （25% ,50% ,75%, 100% ） added into the blood of the untransplanted goats and the cells were labeled with CD34^＋ monoclonal antibody. The region and size of the ＂gate＂ were chosen based on to the distribution of CD34^＋ cells or human cord blood. One human/goat chimera marked with GFP （ MIG goat） was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP 34^＋ cells percentage and DNA contents by flow cytometry. Results CD7 , CD15, CD38, CD45 , CD20, CD34, CD14 and GPA monoclonal antibodies were chosen as the primary antibodies in routine detection by flow cytometry. The size and area of the ＂gate＂ were also defined. 29. 1% （29 100/ 100 000） of the substantial liver cells from the MIG goat expressed GFP. DNA content analysis showed that the GFP 34^＋ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human. Conclusions Flow cytometry is rapid, simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo. The selections of suitable surface antibodies and the ＂gate＂ are very important for detecting human ceils accurately in the human/goat chimerism.

关 键 词：流式细胞术 造血干细胞 移植嵌合体 抗原 表面 

分 类 号：R686[医药卫生—骨科学]