山羊毛囊干细胞分离、培养及鉴定

Isolation,Culture and Identification of Goat Hair Follicle Stem Cells

采用2.4 U/mL Dispase酶消化和机械切割法分离毛囊隆突部,经胰酶(0.5 mg/mL胰酶+0.2 mg/mLEDTA)消化从山羊耳部皮肤分离得到毛囊干细胞,用自制无血清培养基培养,并用形态学观察、细胞生长曲线、克隆形成率及免疫组化染色检测,探讨毛囊干细胞体外分离培养方法及生物学特性。结果表明,所分离细胞具备毛囊干细胞特征:体积较小、表面光滑、立体感强,呈现未分化细胞特征;K19、integrin-β1以及p63免疫组化染色阳性;第3、5、7代干细胞克隆形成率分别为25.6%±2.6%、31.8%±1.9%和27.0%±3.9%,极显著高于对照组角质细胞克隆形成率(P<0.01)。采用配制的无血清条件培养液可使山羊毛囊干细胞体外维持未分化状态并传代至19代。

Hair follicles were isolated from goat ear skin by 2.4 U/mL Dispase digestion and the bulges were separated with special fine needle and digested with 0.5 mg/mL trypsin plus 0.2 mg/mL EDTA. The resultant cells were cultured in self-prepared serum-free conditional medium and studied through morphological observation, cell growth curve and colony-forming efficiency （CFE）, and identified by immunohistochemical staining. The results showed that the cells were slide, small-sized, ball-shped, obviously three-dimensional and undifferentiated, immunohisto- chemically positive to K19, integrin-β1 and p63 staining and so performed stem cells attributes and high CFE. The CFEs of the third, the fifth and the seventh passages were respectively 25.6%4-2.6%, 31.8%±1.9 % and 27.0±3.9%, significantly higher than that of control （P 〈0.01）. And the data proved that the goat follicle stem cells could be subcultured for 19 times in vitro in the serum-free conditional medium self-prepared.