人脂肪组织来源的基质细胞分化为神经元样细胞的体外研究

THE IN VITRO STUDY OF THE HUMAN ADIPOSE TISSUE-DERIVED STROMAL CELLS DIFFERENTIATING INTO THE NEURON-LIKE CELLS

目的探讨人脂肪组织来源的基质细胞(adiposetissue-derivedstromalcells,ADSCs)向神经元样细胞分化的可能性,为神经移植探索新的细胞来源。方法采用胶原酶消化法分离培养成人的ADSCs,含血清的培养基进行培养,胰蛋白酶消化传代,采用第3～9代的ADSCs进行诱导。应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松,诱导其向神经元样细胞和脂肪细胞分化,采用苏丹黑B和免疫细胞化学方法对ADSCs进行鉴定。结果成功培养出ADSCs来源的基质细胞,细胞在体外生长形态类似成纤维细胞,可维持在未分化状态并稳定增殖,体外扩增可达20代。细胞表达波形蛋白和巢蛋白,大部分细胞还表达平滑肌肌动蛋白和β管蛋白;应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松,可诱导ADSCs向神经元样细胞和脂肪细胞分化,其中0.1%～0.2%的细胞分化为神经元样细胞,40%～50%分化为脂肪细胞。分化的神经元样细胞具有典型的神经元形态,并能表达神经元标志物;部分分化的神经元样细胞仍然表达平滑肌肌动蛋白。结论脂肪组织中存在能分化为神经元样细胞的基质细胞,并能克服间充质细胞的限制,分化为神经元样细胞,但这种细胞是否为有功能的神经元,还需深入研究。

Objective To investigate the possibility of the adipose tissue-derived stromal cells （ADSCs） to differentiate into the neuron-like cells and to explore a new cell source for the transplantation related to the central nervous system. Methods Adipose was digested by collagenase, cultured in the fetal bovine serum containing a medium. Trypse was used to digest the cells and the cell passage was performed. The 3rd to the 9th passage ADSCs were used to make an induction. Isobutyl-methylxanthine, indomethacin, insulin, and dexamethasone were used to induce the ADSCs to differentiate into the neuron-like cells and adipocytes, Sudan black B and immunocytochemistry were used to identify the cells. Results A population of the ADSCs could be isolated from the adult human adipose tissue, they were processed to obtain a fibroblast-like population of the cells and could be maintained in vitro for an extended period with the stable population doubling, and they were expanded as the undifferentiated cells in culture for more than 20 passages, which indicated their proliferative capacity. They expressed vimentin and nestin, and characteristics of the neuron precursor stem cells at an early stage of differentiation. And the majority of the ADSCs also expressed the neuron-specific enolase and β Ⅲ-tubulin, characteristics of the neurons. Isobutyl-methylxanthine, indomethacin, insulin, and dexamethasone induced 40 %-50 % of ADSCs to differentiate into adipocytes and 0.1%-0.2 % of ADSCs into neuron-like cells. The neuron-like cells had a complicated morphology of the neurons, and they exhibited a neuron phenotype, expressed nestin, vimentin, neuron-specific enolase and β Ⅲ-tubulin, but some neuron-like cells also expressed the smooth muscle actin （SMA）, and the characteristics of the smooth muscle cells; however, the neurons from the central nervous system were never reported to express this kind of protein. Therefore, the neuron-like cells from the ADSCs could be regarded as functional neurons. Conclusion Our results support the hypothesis that the adult adipose tissue contains the stem cells capable of differentiating into the neuron-like cells, and they can overcome their mesenchymal commitment, which represents an alternative autologous stem cell source for transplantation related to the central nervous system.