由脐血单个核细胞和富集的CD34^＋细胞扩增的造血干／祖细胞的生理功能比较

Hematopoietic repopulating ability of hematopoietic stem/progenitor cells expanded by mononuclear cellsor CD34^＋ cells

目的 探讨由脐血单个核细胞（MNC）和富集的CD34^＋细胞起始扩增所得的造血干／祖细胞在体内植入及造血重建的能力。方法 从人脐血中分离出MNC和CD34^＋细胞，在体外扩增7d。将非肥胖糖尿病型重症联合免疫缺陷型（NOD／SCID）／小鼠分为四组，在接受亚致死剂量铯源照射后，进行细胞移植，实验A组接受由MNC培养得到的CD34^＋细胞和CD34^-细胞；实验B组接受由富集的CD34^＋细胞培养得到的CD34^＋细胞和CD34^-细胞；阳性对照组接受从脐血新鲜分离的CD34^＋细胞和CD34^-细胞；阴性对照组不接受细胞移植，仅输注相同体积的IMDM培养基。6周后处死存活的小鼠，取其骨髓、脾脏和外周血细胞，分别进行细胞表型分析、集落和人特异性基因的检测。结果 经过体外扩增，以富集的CD34^＋细胞起始培养者的细胞总扩增倍数为39．8倍，远高于以MNC为起始细胞者的1．88倍。移植6周后，所有接受细胞移植的小鼠均存活，存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞（CD45^＋细胞）及人源的各系血细胞，实验A组各类细胞的含量稍高于实验B组，且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列。结论与从脐血中新鲜分离的细胞相比，扩增后的造血干／祖细胞的体内植入能力有所下降，以MNC起始扩增的造血干／祖细胞体内植入能力优于以富集的CD34^＋细胞起始扩增者，但二者体内造血重建能力的差异不显著。

Objective To compare the hematopoietic repopulating ability of cord blood hemato-poietic stem/progenitor cells （HS/PC） ex vivo expanded with different initiated cells by Nonobese diabetic/severe combined immunodeficiency （NOD/SCID） mouse model. Methods HS/PC were ex vivo expanded with enriched CD34^＋ cells and mononuclear cells （MNC） derived from umbilical cord blood. The 7-day expanded cells were transplanted into NOD/SCID mice which received sublethal irradiation from a ^137Cs source. Six weeks after transplantation, peripheral blood, spleen and bone marrow cells of the alive mice were harvested for flow cytometric, colony forming cells （CFC） and PCR analyses. Results Enriched CD34^＋ cells presented a higher proliferation potential than MNC in the expansion process. Six weeks after transplantation, all of the mice injected with cord blood cells survived. Human cells （CD45^＋ cells） could be detected in BM and spleen cells of the alive mice, and human CD34, CD3, CD19, CD33 and CD71 antigen all could be detected on these human cells. The human specific Alu sequence could be detected in the DNA obtained from NOD/SCID mice by PCR. Conclusion In vitro expanded cells has a decline of engraftment level in NOD/SCID mice compared with fresh cord blood cells, but there was no significant difference in the multi-lineage engraftment level of them The ex vivo expanded cells initiated with MNC were superior to the cells expanded with enriched CD34^＋ cells in the engraftment level, but different initiated cells had no significant impact on the hematopoietic repopulating ability of expanded HS/PC in vivo.