CAJ | 학술논문

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)的人造血于/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CD+34表面抗体标记来观察人脐血细胞和人CD+34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、CD45CD20CD34CD14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%(29 100/100 000);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.
목적 건립응용류식세포술검측인/산양감합체중인원세포적방법.방법 통과궁내이식적방법장표기유록색형광단백(GFP)적인조혈우/조세포(MIG세포)이식입태산양복강내,종이획득인/산양감합체모형(MIG양).채집MIG양적외주혈,이수십충서항인적항체표기,사선출대인원세포특이성강이여산양세포무혹부유미약교차반응적항체,렬인류식세포의상규검측적수선항체,계이재비이식산양적외주혈중가입불동비례(25%、50%、75%화100%)적인제혈세포,용CD+34표면항체표기래관찰인제혈세포화인CD+34세포적분포,이결정"문"적구역화대소;취MIG양적간장,응용류식세포술검측관류후간실질세포중GFP세포적비례급DNA함량.결과 사선출CD7、CD15、CD38、CD45CD20CD34CD14화혈형당단백A(GPA)등8충단항작위검측감합체중인원세포적수선항체;학정료진행류식세포분석시"문"적대소화구역;MIG양간실질세포중,GFP+세포적비례위29.1%(29 100/100 000);DNA함량측정결과현시MIG양간조직분선획득적GFP+세포주요정현2개봉,기위치여인적쌍봉위치상대응.결론 류식세포술시연구간세포재체내분화、귀소화생물학특정적쾌속、간편、유효적방법;표면항체이급"문"적선택대우준학검측인/산양감합체중인원세포지관중요.
Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 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 monoelonal 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 CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 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+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ 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 cells accurately in the human/goat chimerism.