人脐带间充质干细胞的生物学特性及向神经样细胞分化的研究

目的 研究人脐带华尔通胶来源的间充质干细胞（MSCs）的特性及向神经细胞分化的可能性,为神经移植寻找新的细胞来源。方法 检测人脐带来源的MSCs的细胞表面标记;丹参和B巯基乙醇诱导人脐带来源的MSCs向神经细胞分化,用免疫细胞化学方法对分化和未分化的细胞进行鉴定;半定量逆转录聚合酶链反应（RT—PCR）检测细胞的神经相关基因表达。结果 从人脐带分离、培养的贴壁细胞,体外生长形态类似于成纤维细胞,可以维持在未分化状态稳定增殖,体外增殖超过10代。这类细胞MSCs的表面标记CD29、CD44、CD59、CD105仿呈现高表达,造血细胞表面标记CD14、CD33、CD34、CD27、CD45、CD117和与移植免疫排斥相关的表面标记CD80（137—1）、CD86（B7-2）、CD40、CD40L不表达或低表达。丹参和β巯基乙醇均可诱导人脐带MSCs向神经样细胞分化,分化的细胞表达神经干细胞的标记巢蛋白（Nestin）,神经元的标记类神经微管（β-TubulinⅢ）和神经微丝（NF）,以及神经胶质细胞的标记胶质纤维酸性蛋白（GFAP）。RT-PCR检测证实,经丹参诱导后的MSCs表达神经干细胞相关基因Nestin,诱导前和诱导后的MSCs均表达神经细胞基因Pleiotrophin,诱导后的表达明显增强。结论 人脐带华尔通胶含有丰富的MSCs,易于培养扩增,其表达MSCs的表面标记。不表达或低表达造血细胞和与移植排斥相关的细胞标记。人脐带来源的MSCs能分化为神经细胞,表达神经细胞的相关标记和基因,这种细胞可能成为中枢神经系统细胞移植的一个干细胞来源。

Biological characteristics of human umbilical cord-derived mesenchymal stem cells and their differentiation into neurocyte-like cells

OBJECTIVE: To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells. METHODS: The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR. RESULTS: A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed. CONCLUSION: The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.