大鼠骨髓间质多潜能成年祖细胞体外培养和神经分化潜能的研究

Rat bone marrow muitipotent adult progenitor cells expand anddiffetentiate into neuron-like cells in vitro

目的:探讨大鼠骨髓间质中多潜能成年祖细胞(muhipotent adult progenitoi-cells or MAPCs)的体外培养与神经分化的方法,观察其增殖和分化特点。方法:采用Ficoll-Paque液(1.077g/L)和免疫微磁珠从大鼠双侧股骨和胫骨骨髓中分离与纯化出多潜能成年祖细胞,体外扩增,分别采用含碱性成纤维生长因子(bFGF)和脑源性神经营养因子(BDNF)、成纤维生长因子-8(FGF-8)等试剂的无血清L-DMEM诱导MAPC分化为神经元。应用免疫荧光化学技术和RT-PCR等方法对分化细胞进行鉴定。结果:多潜能成年祖细胞在休外扩增可超过50代,细胞形态和神经分化能力没有发生变化。在碱性成纤维生长因子的诱导下,MAPC形态转变为典型的神经样细胞,神经元特异性烯醇化酶(NSE)表达阳性。继续用BDNF、FGF-8持续诱导,MAPC可分化为更加成熟的神经样细胞,微管相关蛋白-2(MAP-2)表达阳性;RT-PCR显示诱导出的神经样细胞,酪氨酸羟化酶(TH)、多巴胺β-羟化酶(DBH)mRNA表达水平明显升高。结论:建立了多潜能成年祖细胞体外分离、培养的条件,探索了多潜能成年祖细胞部分生物学特性,多潜能成年祖细胞在体外可以持续增殖并可诱导分化为较成熟的神经元样细胞,它可为多潜能成年祖细胞的临床应用提供材料。

Objective: To investigate expansion and differentiationof rat bone marrow multipotent adult progenitor cells (rMAPCs) intoneuron-like cells in vitro. Methods:The bone marrow were separatedfrom femurs and tibias of rats, BM mononuclear cells obtained by Ficoll-paque density gradient centrifugation, depleting of CD45+ andglycophorin-A-positive (GlyA+)cells by means of micromagneticbeads, the eluted ceils were rMAPCs, rMAPCs expanded inL-DMEM culture medium supplemented with 10% FCS, 100ng/mlleuxaemia inhibitory factor(LIF), rMAPCs were induced to dif-ferentiateinto neuron-like cells by bFGF/L-DMEM or by bFGF , BDNF, FGF - 8/L-DMEM. At the same time rMAPCs were cultured in L-DMEM incontrol group. Neuron-specific enclose(NSE) and Microtubule-associatedprotein - 2(MAP-2) were detected by immunofluorescence method, Levelof TH and DBH mRNA were detected by RT-PCR. Results: rMAPCsexpanded for more than 50 cell doublings in L-DMEM culture mediumsupplemented with 10% FCS, 100ng/ml LIF, indicating their differentiatedcapacity, rMAPCs exhibit neuronal phenotype, expressing a positiveNSE induced by bFGF/L-DMEM and more mature neuronal phenotypeinduced by bFGF, BDNF, FGF-8 / L-DMEM, expressing a positiveMAP-2,levels of TH and DBH mRNA were upregulated significantly. rMAPCs were not induced to differentiate into neuron-like cells incontrol group. Conclusions: The best condition for isolation and cultureof rat bone marrow MAPCs were successfully established and somebiological features were observed, rMAPCs could expanded and beinduced to differentiate into neuron-like cells in vitro. It found a base forfurther investigation and using of rMAPCs in clinic.