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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,提示hAMCs具有向神经元分化的潜能
Objective: To investigate the neurobiological characteristics of cultured human amniotic mesenchymal cells (hAMCs) in vitro. Methods: MTS assay, BrdU incorporation and proliferating cell nuclear antigen (PCNA) immunofluorescence staining were used to investigate the proliferative activity of hAMCs cultured in vitro. The expression of STRO-1, Nestin (PSA-NCAM), β-tubulin-Ⅲ (TH) and neural differentiation-related proteins in hAMCs were detected by immunocytochemistry (math-1, mash-1) expression. Results: MTS assay showed that hAMCs proliferated most rapidly on day 6-8 after inoculation, and maintained viable cells on days 8-24. Immunofluorescence staining of BrdU and PCNA showed that there were abundant BrdU and PCNA in hAMCs cultured in vitro Positive hMSCs were cultured in vitro. The hMSCs cultured in vitro expressed mesenchymal stem cell-specific markers STRO-1 and Vimentin as well as neural stem cell markers Nestin and PSA-NCAM. Neuron-specific markers β-tubulin-Ⅲ and TH, as well as the expression of math-1 and mash-1 related to neuronal differentiation. There were TH / BrdU and β-tubulin-Ⅲ / BrdU double positive cells in hAMCs cultured in vitro. CONCLUSION: hAMCs cultured in vitro express specific marker proteins of mesenchymal stem cells, neural stem cells and neurons and also have proliferative activity: hAMCs express neuronal differentiation-related proteins, suggesting that hAMCs have the potential to differentiate into neurons