CD48表达可作为区分造血干细胞分裂方式的活性标志

Expression of CD48 as A Live Marker to Distinguish Division of Hematopoietic Stem Cells

造血干细胞在细胞分裂时具有维持自我更新和产生定向分化的能力,其依赖于三种细胞分裂方式,一个干细胞产生两个新的干细胞(对称的更新分裂)、两个分化细胞(对称的分化分裂)或者一个干细胞和一个分化细胞(不对称分裂)。本研究旨在探索一种高效、稳定的区分造血干细胞分裂方式的方法。前期研究表明,Numb蛋白的分布情况可以作为许多细胞辨别分裂方式的标志,本研究利用免疫荧光技术检测Numb蛋白在小鼠分裂期CD48-CD150+LSK细胞中的分布情况,探索Numb蛋白与中心体的关系。由于CD48表达阳性标志着细胞失去了骨髓重建能力,因此本研究把CD48的表达作为区分造血干细胞自我更新或定向分化的一个活性标志。从小鼠全骨髓细胞中分选出CD48-CD150+LSK细胞,培养体系中加入自行制备的AF 488-conjugated anti-CD48抗体,培养3 d后共聚焦荧光显微镜观察及流式细胞分析是否存在AF488+细胞及其所占比例,然后二次分选AF488+和AF488-细胞进行细胞集落形成实验(colony forming cell assay,CFC)与细胞增殖能力比较。结果表明:Numb蛋白在处于分裂期的CD48-CD150+LSK细胞中对称或不对称分布在细胞分裂平面两侧,这提示Numb蛋白染色可以作为一种区分造血干细胞分裂方式的方法。此外,CD48-CD150+LSK细胞在含有自行制备的AF488-conjugated anti-CD48抗体的培养体系中培养3 d后产生约40%AF488+细胞,共聚焦荧光显微镜统计的阳性细胞比例与流式细胞分析检测结果一致。二次分选AF488+和AF488-细胞,AF488+细胞群的集落形成能力显著高于AF488-细胞群(P<0.05),AF488-细胞群的增殖能力显著高于AF488+细胞群(P<0.05)。结论:CD48表达作为细胞干性丢失的活性标志,可区分造血干细胞的分裂方式。该方法与Numb蛋白染色方法相比,具有用于活细胞的优势,它为后续的细胞培养与细胞移植等研究工作提供了更大的便利。

Hematopoietic stem cells are capable of self-renewal or differentiation when they divide. Three types of cell divisions exist. A dividing stem cell may generate 2 new stem cells （symmetrical renewal division ）, or 2 differentiating cells （symmetrical differentiation division）, or 1 cell of each type （asymmetrical division）. This study was aimed to explore an efficient and stable method to distinguish the way of cell division in hematopoietic stem cells. Previous studies showed that the distribution of Numb in a cell could be used to distinguish the type of cell division in various kinds of cells. Therefore, the distribution of Numb protein was detected by immunofluorescence in mitotic CD48- CD150＋ LSK cells of mice exploring the relationship between Numb protein and centrosomes. Since CD48 positive marks the HSC that have lost the ability to reconstitute the blood system in mice, CD48 marker could be used to distinguish cell fate decision between self-renewal and differentiation as a living marker. In this study, the CD48- CD150 ＋ LSK cells were sorted from bone marrow cells of mice and the cells were directly labeled with Alexa Fluor （AF） 488-conjugated anti-CD48 antibody in living cultures. After 3 days, the percentage of AF488 ＋ cells was evaluated under microscope and by FACS. Then colony forming cell assay （CFC） was perfomed and the ability of cell proliferationwere compared between AF488 ＋ and AF488 - cells. The results showed that Numb could be used to distinguish different cell division types of hematopoietic stem cells, which was symmetrically or asymmetrically segregated in mitotic CD48 - CD150 ＋ LSK cells. The self-labeled fluorochrome could be detected both by FACS as well as microscope. There were about 40% AF488 ＋ ceils after 3 day-cultures in medium titrated with self-labeled AF 488-conjugated anti-CD48 antibody, and the results were consistent between confocal fluorescence microscopy and flow cytometry analysis. The colony forming ability of AF488 ＋ cells was significantly higher than that of AF488 - cells （ P 〈 0.05 ）. The proliferation ability of AF488- cells was also significantly higher than AF488 ＋ cells （P 〈0.05）. It is concluded that the expression of CD48 can distinguish cell division of hematopoietic stem ceils and can be used as a live marker for the loss of stemness. In comparison with the Numb protein staining, this method can be used in living cells, thus provides greater convenience for subsequent cell culture studies and cell transplantation experiments.