Bcr/Abl融合基因阳性、Flk1+CD34-慢性粒细胞性白血病干细胞的分离纯化及其生物学特性的研究

目的分离慢性粒细胞白血病(ChronicMyelogenousLeukemia,CML)患者骨髓中表达BCR/ABL肿瘤基因的细胞亚群,并研究其生物学特性。方法淋巴细胞分离液分离CML患者骨髓单个核细胞,免疫磁珠分选CD45-、GlyA-及CD34-细胞,极限稀释法获得单克隆细胞,流式细胞术鉴定其免疫表型,体外定向诱导分化检测其干细胞特性,并检测其Bcr/Abl融合基因的表达情况。结果CML患者骨髓源单克隆扩增细胞不表达造血细胞和内皮细胞的特征性表型,但Bcr/Abl融合基因阳性;诱导后的细胞能同时向造血细胞及血管内皮细胞分化。结论Bcr/Abl融合基因的产生至少发生在比造血干细胞更为早期的血液血管干细胞水平上,即CML至少起源于血液血管干细胞。

The Primary Study of biological characteristics and isolation of Bcr/Abl+、Flk1+CD34-stem cells of Chronic Myelogenous Leukemia

Objective To isolate subgroup cells carrying Bcr/Abl fusion gene from the bone marrow of CML patients, and study the biological characteristics of them. Methods Mononuclear cells from bone marrow of Ph patients with CML were separated by a Ficoll-Paque gradient centrifugation, using magnetic-activated cell separation (MACS) filtered the CD45-, GlyA-, and CD34- cells, mono-clonal cells were harvested by limiting dilution; then identified the immunophenotype by fluorescence-activated cell sorter (FACS), targeting differentiation in vitro to detect the characteristics of the stem cells, furthermore, examined the Bcr/Abl fusion gene expression of them. Results The mono-clonal proliferation cells from the bone marrow of patients with CML do not express the phenotype of hematopoietic cells and endothelial cells, however, they can express the Bcr/Abl fusion gene; the cells after inducement could give rise to both hematopoietic cells and endothelial cells at the same time. Conclusion These findings provide the direct evidence that rearrangement of Bcr/Abl gene might happen at least more primitive than CD34 CD38- cells, i.e., it may occur at the level of putative hemangioblasts. That is, CML arises at the level of hemangioblasts.