培养前后脐血CD34+细胞在NOD-SCID鼠体内造血重建功能比较.docVIP

培养前后脐血CD34+细胞在NOD/SCID鼠体内造血重建功能比较 　 作者：杨施, 蔡海波, 金慧丽, 谭文松 【摘要】 目的: 以NOD/SCID小鼠为模型, 经半致死剂量照射后输注新鲜或培养后的造血细胞, 以比较培养前后脐血CD34+细胞的造血重建功能。方法: 从新鲜脐血中分离单个核细胞（MNC）, 采用干细胞因子（SCF）、 血小板生成素（TPO）、 Flt3配体（FL）、 白细胞介素3（IL3）和白细胞介素6（IL6）细胞因子组合体外培养14 d。通过MiniMACS免疫磁性吸附柱从新鲜或培养后的MNC中分离CD34+细胞, 4×105个CD34+细胞和5×106CD34-细胞混合后通过尾静脉输注入NOD/SCID小鼠中。饲养过程中动态观察外周血象恢复情况, 6周后检测小鼠骨髓和脾脏细胞中人源细胞及各系造血细胞的含量。结果: 体外培养MNC 14 d后, 总细胞扩增了1.78倍; 细胞移植6周后, 输注新鲜和培养后造血细胞的小鼠均存活, 在小鼠骨髓和脾脏中均可检测到人源细胞及各系人源血细胞和人特异ALU基因序列, 小鼠外周血象恢复到辐照前水平。培养后CD34+细胞在小鼠体内的植入水平与新鲜CD34+细胞的相近, 而其各系人源血细胞的含量高于新鲜CD34+细胞。 结论: 体外培养14 d后的CD34+细胞仍保持了体内植入和重建造血的能力, 且其多系造血重建能力优于新鲜CD34+细胞。 【关键词】 CD34+细胞; 造血重建; NOD/SCID小鼠 ［Abstract］ AIM: To study the hematopoietic reconstitution ability of fresh and cultured cord blood CD34+ cells by sublethally irradiated NOD/SCID mice. METHODS: Mononuclear cells (MNC) were separated from cord blood and then cultured with stem cell factor (SCF)+ flt3 ligand (FL)+thrombopoietin (TPO)+ interleukin3 (IL3)+ IL6 for 14 d. CD34+ cells were isolated from fresh or cultured MNC using miniMACS magnetic separation system. Then 4×105 CD34+ cells together with 5×106 CD34- cells were injected into NOD/SCID mice. After transplantation, the dynamics of hematopoieitc recovery in the mice were measured. After 6 weeks, bone marrow (BM) and spleen samples were obtained from mice for detecting human cells. RESULTS: The number of total cells increased 1.78fold after 14 d of culture. All recipients received fresh and cultured CD34+ cells survived. Human cells, human lineage cells and human ALU sequence could be detected in BM and spleen. The peripheral blood counts of all transplanted mice recovered. The engraftment levels of cultured CD34+ cells were similar to those of fresh CD34+cells but the percentage of human lineage cells was higher than that of fresh CD34+ cells. CONCLUSION: CD34+ cells cultured for 14 d still preserved the hematopoietic reconstitution ability and showed better multilineage reconstitution ability than fre