体外培养人羊膜间充质细胞的神经生物学特点

The neurobiology characteristics of human amnion mesenchymal cells culture in vitro

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掺人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。

Objective： To evaluate the neurobiological features of human amnion mesenchymal cells （hAMCs） in vitro. Method： CellTiter 96 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. Immunoeytoehemistry 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. Immunofluoreseence demonstrated some of hAMCs were positively stained by anti-BrdU and anti-PCNA.② Cultured hAMCs were able to express mesenehymal 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 ceils, 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.