人类皮肤来源前体细胞的表型和特征

背景：研究证实，从人类的皮肤中成功分离出皮肤源性前体细胞并在体外长期培养，可分化为神经元、胶质细胞、平滑肌细胞以及具有周围神经元表型的细胞和许旺细胞。目的：进一步证实人皮肤组织来源的多能干细胞的培养方法和多方向分化能力，尤其向神经细胞、成骨细胞分化的可能性。方法：采用胰蛋白酶消化法培养人皮肤组织来源的前体细胞，应用免疫细胞化学的方法对细胞进行鉴定。取三四代的细胞分别进行神经元诱导分化、成骨细胞诱导分化、yonKossa法钙染色、软骨细胞诱导分化、甲苯胺蓝染色、免疫组织化学染色及苏丹黑染色。观察皮肤来源的前体细胞巢蛋白、波形蛋白、βⅢ管蛋白、S100及Ⅱ型胶原的表达。结果与结论：用胰蛋白酶消化法培养出人皮肤组织来源的前体细胞，增殖聚集能够形成悬浮的细胞球，在培养的不同时间都可检测到巢蛋白阳性的细胞。培养的全部细胞表达波形蛋白，在贴壁的细胞中也能检测到βⅢ管蛋白的表达。利用丹参诱导皮肤源性前体细胞向神经元样细胞分化，表达神经细胞的标志物。应用成骨细胞诱导，yonKossa染色发现培养皿中有黑色的钙化结节产生，说明皮肤源性前体细胞能够分化为成骨细胞；将皮肤源性前体细胞向软骨细胞诱导，甲苯胺蓝染色强阳性，部分细胞表达Ⅱ型胶原，证明分化的细胞中含有软骨细胞。实验结果证实，皮肤中存在的多能干细胞能够分化为成骨细胞、软骨细胞、许旺细胞和少突胶质细胞。

Phenotypes and characteristics of human skin-derived precursors

BACKGROUND： Human skin-derived precursors can be cultured for a long term in vitro, and differentiated into neurons, glial cells, smooth muscle cells, Schwann cells and cells with peripheral neurons phenotype. OBJECTIVE： To investigate the culture conditions and multiple differentiation capacity of multipotential stem cells from human skin, especially the potentials of differentiating into neurons and osteoblasts. METHODS： Human skin-derived precursor cells were cultured with trypsin digestion method, and identified with immunocytochemistry. Cells at passages 3-4 were induced to differentiate into neurons and osteoblasts, and underwent von Kossa staining protocol for calcium, chondrocyte induction, toluidine blue staining, immunohistochemical staining and Sudan black staining. The expression of nestin, vimentin, ~lll-tubulin, $100 and collagen II in the human skin-derived precursors was detected. RESULTS AND CONCLUSION： The human skin-derived precursor cells cultured with trypsin digestion method could proliferate and form suspending spheres, and nestin positive cells were detected at any time point of theculture. All the cultured cells expressed vimentin, and some adherent cells expressed 13111-tubulin. Human skin-derived precursor cells were induced with Salvia miltiorrhiza to differentiate into neuron-like cells, and expressed marker of nerve cells. Skin-derived precursors could be induced to differentiate into osteoblasts and von Kossa staining displayed black calcified nodules in the culture dish. Skin-derived precursors could also be induced to differentiate into chondrocytes, and toluidine blue staining was strongly positive, and some cells expressed collagen If, which suggested that, the differentiated cells contained chondrocytes. Experimental findings indicate that, skin contains multipotential stem ceils that are capable of differentiating into osteoblasts, chondrocytes, Schwann cells and oligodendrocytes.