羊驼毛囊干细胞分离、培养与鉴定

Isolation,culture and identification of alpaca hair follicle stem cells

采用0．2％中性蛋白酶Ⅱ和0．25％胰酶：0．02％EDTA（1：1）“两步酶”消化法从羊驼背部皮肤分离得到羊驼毛囊干细胞。用无血清角质细胞培养基（K-SFM）培养进行形态学观察、细胞生长曲线克隆形成率及免疫组化染色检测。结果显示，细胞生长曲线表明，不同代次毛囊干细胞接种前3d生长缓慢，4～6d进入倍增期，有无限增殖的趋势，所分离的羊驼毛囊干细胞具备毛囊干细胞特征（体积小、立体感强），呈现未分化特征；CK19、CK15、81-integrin和CD34免疫组化染色阳性；第3、5、7、9代克隆形成率分别为（32．7±2．27）％、（47．0±3．46）％、（46．3±3．18）％和（43．3±3．76）％。结果表明，用“两步酶”消化法成功分离获得羊驼毛囊干细胞，无血清角质细胞培养基（K—SFM）可使羊驼毛囊干细胞体外维持未分化状态并传代至12代。

The sample was taken from a piece of alpaca back skin,and hair follicle stem cells were isolated by digested 0.2 % dispase enzymes and 0.25 % trypsin, which were cultured in K-SFM se- rum-free conditional medium. The morphological observation, cell growth curve and colony-form- ing efficiency （CFE）,and identified by immunohistochernical staining. The growth curve of ceils showed that different passages of hair follicle stem cells grew slowly at 3-day post inoculation,and went into double time at 4-6 days which had an infinite proliferation trend. And with the feature of small-sized, obviously three-dimensional and undifferentiated, it is the same with that of hair follicle stem cells. CK19, CK15, integrin-β1 and CD34 appeared as positive with immunohistochemistry,and the CFEs of the third, fifth, and seventh passages were （32.7±2.27）%, （47.0± 3.46） %, （46.3±3.18）% and （43.3%3.76）% ,respectively. Hair follicle stem cell s were separated successfully with ＂two steps enzyme＂ digestion method, which could be subcultured for 12 times in vitro in the serum-free keratinocyte medium（K-SFM）.