人羊膜组织细胞的神经干细胞特性形态学研究

The morphologic study of phenotypes expression of neural stem cell from human amniotic cells

目的:利用形态学研究方法,探讨神经干细胞特异性标志蛋白的表达和增殖分化特性,鉴定人类羊膜组织中神经干/前体细胞的存在。方法:利用免疫组织化学法,检测人类羊膜组织和培养人类羊膜细胞中nestin、musashi-1、vimentin和PSA-NCAM等神经干细胞特异性标志蛋白的表达,利用BrdU掺入实验鉴定羊膜组织来源神经干/前体细胞的增殖分化能力。结果:免疫组织化学检测证实人羊膜组织表达nestin、musashi-1、CD133和vimentin等神经干细胞特异性标志蛋白,主要分布于羊膜组织的间质层。培养羊膜细胞的免疫荧光化学染色显示nestin、musashi-1、vimentin和PSA-NCAM阳性细胞的存在。BrdU掺入的组织形态学实验显示培养羊膜细胞中存在BrdU标记阳性细胞,其中具有BrdU与nestin、musashi-1、vimentin双标阳性细胞,显示羊膜细胞中存在具有增殖活性神经干细胞标记蛋白表达阳性细胞;而BrdU与β-tubullinIII、TH和GFAP双标阳性细胞的存在,表明神经元和胶质细胞是由培养羊膜细胞增殖分化的。结论:羊膜组织中存在神经干细胞特异性标记蛋白Nestin、Musashi-1、CD133、PSA-NCAM和Vimentin表达阳性细胞,BrdU掺入的组织形态学结果不仅证实羊膜细胞中Nestin、Musashi-1、Vimentin阳性细胞具有增殖活性,而且其中存在已分化的神经元和胶质细胞,提示羊膜组织细胞内存在神经干/前体细胞。

Objective： Amniotic membrane which formed at the early stage of embryonic development, consists of epithelial cells and mesenchymal cells and possesses the potency to differentiate into multi-germinal layer cells. Here we investigate the expression of specific protein markers of the neural stem cells （NSCs）, the proliferation and differentiation of the amniotic cells by utilizing the morphology methods, identifying the neural stem/precursor ceils in human amniotic membrane. Method： The expression of specific protein markers of neural stem cells were detected by immunocytochemistry in human amniotic membrane and cultured amniotic ceils respectively. The proliferation of neural stem/precursor cells derived from human anmiotic cells were tested by BrdU incorporation. Result： Pluripotent neural stem cell specific makers were detected in the human amnion membrane, especially in mesenchymal layer. In addition, several neural stem cell specific markers including Nestin, Musashi-1, Vimentin and PSA-NCAM positive cells were identified in the cultured anmiotic cells by immunofluorescence staining. BrdU incorporation test showed that BrdU positive cells existed in cuhured human amniotic cells, and double staining of Nestin＋/BrdU＋, Musashi-l＋/ BrdU＋, Vimentin＋/ BrdU＋ and PSA-NCAM＋/ BrdU＋ suggested that proliferative cells expressing specific protein markers of the neural stem cells presented in cuhured amniotic cells. Conclusion： Positive cells of neural stem cells specific protein markers including Nestin,Musashi-1, CD133, PSA-NCAM and Vimentin existed in human amnion membrane. BrdU incorporation test showed the existence of both the proliferative cells expressing Nestin,Musashi-1 and Vimentin, and differentiated neuron and gila cells, which indicated neural stem / precursor cell presented in human amniotic membrane.