慢性粒细胞性白血病骨髓间充质干细胞的分离纯化及其功能特性的研究

目的 分离、纯化慢性粒细胞性白血病（CML）患者骨髓间充质干细胞（MSC）,并对MSC进行功能、特性鉴定。方法 获取CML患者的骨髓MSC,应用极限稀释法获取单克隆来源的MSC,并在低血清培养液中培养和扩增;流式细胞术检测MSC免疫表型和细胞周期;通过油红O染色、Von Kossa染色和Western印迹检测MSC向脂肪、骨和神经细胞的分化。电镜检测CML患者骨髓MSC的超微结构;通过软琼脂培养法和裸鼠接种,确定CML来源的MSC是否存在致瘤性。结果 MSC在相应的诱导条件下可以向骨、脂肪和神经细胞分化。CML来源的MSC在倒置显微镜下为梭形,表达CD29、CD44、CDl05,而CDllb,CD31、CD34、CD45、HLA—DR均为阴性。CML来源的MSCBCR／ABL融合基因阴性,不具备体内和体外致瘤性。结论CML患者骨髓中存在具有多向分化能力的MSC,该细胞群体不具备体外和体内致瘤性,表现为正常MSC的生物学特征。

Isolation and identification of chronic myelogenous leukemia bone marrow mesenchymal stem cells and their functional characteristics

OBJECTIVE: To isolate and culture bone marrow mesenchymal stem cells (MSCs) from chronic myelogenous leukemia (CML) patients and examine their functional characteristics. METHODS: Bone marrow was extracted from the anterior superior iliac spines of 21 patients with CML. MSCs were isolated and cultured. Single colony derived MSCs were harvested by limiting dilution. The cell cycle and immunophenotype of the expanded clonal MSCs were detected by fluorescence-activated cell sorter (FACS). Different agents were used to induce the MSCs to differentiate into osteocyte, adipocyte and neural cells. Von Kossa staining, oil-red staining, and Western blotting was used to examine the ability of differentiation. PCR was used to detect the expression of BCR/ABL gene. The ultrastructure of the CML derived MSCs was observed with electron microscopy. Sixteen BALB/c nude mice were randomly divided into 2 equal groups to be inoculated with HL60 cancer cells and CML derived MSCs to observe the tumorigenicity. MSCs were cultured in soft agar for 2 weeks to observe the clone growth. RESULTS: Fibroblast-like, positive in CD29, CD44, and CD105, and negative in CD116, CD34, CD48, and HLA-DR, the CML derived MSCs could differentiate into osteocyte, adipocyte and neural cells. CML derived MSCs showed normal karyotype and ultrastructure, they did not express BCR/ABL gene. After 2 weeks' culture no clone was formed from the MSCs. Four weeks after tumors were shown in 6 of the 8 mice inoculated with HL60 cells, and no tumor was seen in the mice inoculated with MSCs. CONCLUSION: Able to differentiate into different types of cell and without tumorigenicity, MSCs from the bone marrow of CML patients have the potentiality in clinical application.