体外培养人羊膜间充质细胞的神经生物学特点

目的：探讨体外培养人羊膜间充质细胞（hAMCs）的神经生物学特征。方法：应用MTS分析法、BrdU掺入法和增殖细胞核抗原（PCNA）的免疫荧光染色，探讨体外培养hAMCs的增殖活性。利用免疫细胞化学法检测hAMCs 中间充质细胞标记（STRO-1、Vimentin）、神经干细胞标记蛋白（Nestin、PSA-NCAM）、神经细胞标记蛋白（β-tubulin-Ⅲ、TH）和神经分化相关蛋白（math-1、mash-1）的表达。结果： MTS分析法显示hAMCs在接种后第6—8天增殖速度最快，第8—24天活细胞数基本保持稳定；BrdU和PCNA的免疫荧光染色结果显示体外培养hAMCs中具有大量BrdU和PCNA阳性细胞存在；体外培养的hAMCs表达间充质干细胞特异性标记物STRO-1和Vimentin，也表达神经干细胞标记物Nestin和PSA-NCAM，同时还可见神经元特异性标记物β-tubulin-Ⅲ和TH，以及神经元分化相关蛋白math-1和mash-1的表达；体外培养hAMCs中存在有TH/BrdU和β-tubulin-Ⅲ/BrdU双阳性细胞。结论：体外培养的hAMCs表达间充质干细胞、神经干细胞、神经元的特异性标记蛋白，同时具有增殖活性；hAMCs表达神经元分化相关蛋白，提示hAMCs具有向神经元分化的潜能。

The neurobiology characteristics of human amnion mesenchymal cells culture in vitro

Objective: To evaluate the neurobiological features of human amnion mesenchymal cells (hAMCs) in vitro. Method: CellTiter 96~(R) AQueous One Solution Cell Proliferation Assay (MTS) , BrdU incorporation and detection of proliferating cell nuclear antigen (PCNA) were applied to evaluate the self-proliferation ability of hAMCs.Immunocytochemistry was used to detect the expressions of markers characteristic of mesenchymal stem cell (STRO-1 and Vimentin), neural stem cells (Nestin and PSA-NCAM), neural cells (β-tubulin-Ⅲ and TH), and the proteins related to neural differentiation (math-1, mash-1). Result:①MTS analysis showed that from the 6th d to the 8th d the amount of live hAMCs grew fast; from the 8th d to the 24th d, the amount of live hAMCs was relatively stable. Immunofluorescence demonstrated some of hAMCs were positively stained by anti-BrdU and nti-PCNA. ② Cultured hAMCs were able to express mesenchymal stem cell markers STRO-1 and Vimentin, neural stem cell markers Nestin and PSA-NCAM, neuron markers β-tubulin-Ⅲ and TH and neural differentiation related protein math-1 and mash-1.③Some of hAMCs coexpressed TH/BrdU and β-tubulin-Ⅲ/BrdU. Conclusion: In vitro, hAMCs not only express phenotype of mesenchymal stem cells, neural stem cells and neurons, but also have the capacity of self-proliferation; hAMCs express the proteins related to neural differentiation and have the potential to differentiate into neural-like cells.