慢性粒细胞性白血病患者间充质干细胞的分离、纯化及向神经元样细胞分化的研究

目的研究慢性粒细胞性白血病（CML）骨髓间充质干细胞（MSCs）的分离、纯化、扩增以及在体外向神经元样细胞定向分化的能力。方法获取CML患者的骨髓MSCs，采用低血清培养液培养，瑞士染色观察其形态；流式细胞仪检测其免疫表型和细胞周期；逆转录-聚合酶链反应（RT—PCR）检测CML特异性表达的bcr／abl基因；分析骨髓MSCs染色体核型。全反式维甲酸（ATRA）诱导MSCs在体外分化为神经元样细胞，倒置相差显微镜观察神经元样细胞的形态，免疫组织化学染色法和Western blot法检测神经丝蛋白（NF）和神经元烯醇化酶（NSE）的表达。结果CML来源的骨髓MSCs呈纤维样贴壁生长，体外培养易扩增；表达相关抗原CD29、CD44、CD105，不表达CD31、CD13、CD34、CD45、HLA—DR；不表达bcr／abl融合基因，且具有正常的核型；不同扩增代数的MSCs在诱导剂的作用下呈现典型的神经元样细胞形态，NF、NSE呈阳性反应。结论CML患者骨髓中可以分离培养MSCs，它具有体外大量扩增并保持低分化状态的特性和定向分化为神经元样细胞的能力，是一种可用于神经系统疾病治疗的种子细胞。

Isolation and in vitro Differentiation into Neuron Like-cells of Bone Marrow Derived Mesenchymal Stem Cells in Patients with Chronic Myelogenous Leukemia

Objective To study the characteristics of bone marrow derived mesenchymal stem cells (MSCs) and examine their neuron differentiation capacity in vitro in chronic myelogenous leukemia. Methods Mononuclear cells from bone marrow in the patients with chronic myelogenous leukemia were obtained and cultured in medium to obtain MSCs. The growth curve, cell cycle and immunophenotype were investigated. All-trans-retinoic-acid (ATRA) was used to induce the differentiation of MSCs into neuron-like cells. The differentiated cells were observed under phase-contrast microscopy, and neuron-specific enolase (NSE) and neurofilament (NF) were detected by immunocytochemistry and Western blot. The chimeric oncogene bcr/abl and Ph chromosome were detected in CML-MSCs, CML derived hematopoietic cells and K562 cells. Results Chronic myelogenous leukemia derived MSCs displayed a fibroblast-like morphology adhering to the culture plate. FACScan flow cytometer also showed that the cells expressed several MSCs-related antigens such as CD29, CD44 and CD105, while CD13, CD31, CD45, CD34 and HLA-DR were negative. Under suitable conditions, these MSCs had ability of neuron differentiation. In CML-MSCs, the expression of bcr/abl and Ph chromosome was not detectable. Conclusion We isolated and cultured a population of cells with multipotent stem cells from CML bone marrow. They could proliferate and differentiate into neuron-like cells in vitro. MSCs can be used as an alternative source of cells for neural disease therapy.