不同年龄人真皮多能干细胞的分离及体外分化特点

Isolation and differentiation characteristics of dermal multipotent stem cells from humans of different ages culured in vitro

目的研究不同年龄人真皮多能干细胞的分离、鉴定及体外分化特点。方法切取胎儿、患儿、中青年患者、老年患者正常皮肤标本（1cm×1cm），采用干细胞培养基筛选原代细胞克隆并记录其数量和直径；检测各种标本来源细胞克隆的体外分化情况；行单个细胞克隆分化分析，并检测其相关蛋白的表达。结果胎儿、患儿、中青年患者、老年患者正常皮肤原代培养获得的细胞克隆数分别为（20．1±2．5）×10^2、（15．8±5．7）×10^2、（10．8±1．3）×10^2、（6．2±1．4）×10^2个，两两比较，差异有统计学意义（P＜0．01）；克隆直径分别为（83±12）、（55±10）、（46±12）、（42±8）μm，胎儿＞患儿＞中青年及老年患者（P＜0．05）。各种细胞克隆均能向神经元细胞、神经胶质细胞、平滑肌细胞和脂肪细胞分化，其中胎儿来源的细胞克隆有向神经元细胞分化的优势。培养1个月，胎儿、患儿、中青年患者、老年患者单个细胞克隆形成率依次为41．1％、25．5％、17．7％、15．2％；单个细胞克隆亦能同上多向分化，同时表达巢蛋白、纤维连接蛋白、原癌基因c-mye、信号转导和转录激活因子3、端粒体逆转录酶。结论采用干细胞培养基能够有效分离不同年龄人真皮中具有克隆增殖和多向分化潜能的干细胞，年龄对其的数量、增殖和分化有明显影响。

Objective To study isolation, identification and differentiation characteristics of dermal multipotent stem cells from human of different age in vitro culture. Methods Skin samples（ 1 cm× 1 cm） were harvested from fetus, infant, adult and elderly. The original clones were screened in stem cell medium. The diameter and number of clones were recorded. Analysis of each clone and determination of the expression of various related proteins were carried out. Results The number of suspended clones from normal skins of fetus, infant, adult and the elderly were （20.1 ±2.5） × 10^2, （ 15.8 ±5.7） × 10^2, （ 10.8 ± 1.3） × 10^2, （6.2 ± 1.4） × 10^2, respectively （ P 〈 0.01 ）, while the diameter of the clones from them were （ 83 ± 12） μm, （55 ±10）μm, （46 ± 12）μm, （42±8）μm, respectively （P 〈0.05）. Cloned cells from fetus, infant, adult and elderly could differentiate into neuron cell , neuroglia cell, smooth muscle cell, and adipocyte. The clones from fetus were inclined to differentiate into neuron cells, but those from infant were indined to differentiate into neuroglia cells, and those from adult and elderly were inclined to differentiate into adipocytes. After 1 month of culture, the clone forming rate of the cells from fetus, infant, adult and elderly were 41.1% ,25.5% , 17.7% , 15.2% , respectively. The individual clone cells also showed ability of multidirection differentiation. Nestin, fibronectin, c-Myc, STAT3 and hTERT protein were expressed in all clones. Conclusion Muhipotent stem cells with multi-direction differentiation and proliferation can be efficiently isolated from dermis of human of different age in stem cell culture medium. The number, proliferation and differentiation of dermal multipotent stem cells can be affected by age.