细毛羊毛囊干细胞分离培养方法比较研究

Comparative Study on Isolation and Culture Methods of Hair Follicle Stem Cells in Fine-wool Sheep

试验旨在对细毛羊毛囊干细胞的分离培养方法进行初步研究,建立细毛羊毛囊干细胞体外培养体系。采用两步酶消化法、机械分离法和两步酶消化法+机械分离法3种方法分离培养细毛羊毛囊干细胞,通过测定毛囊干细胞的数量、克隆形成率及毛囊干细胞的增殖能力等综合评估3种培养方法的优劣,寻求既能方便获得大量毛囊干细胞,又能减少其分化的理想培养条件。倒置显微镜下观察3种培养方法获得的细胞均为铺路石状,均表达角蛋白K19和整合素β1,表明成功获得细毛羊毛囊干细胞,建立体外培养体系。采用"两步酶消化法和机械分离法"相结合的方法获得毛囊干细胞体外培养效果最优。

The experiment was a preliminary study on fine-wool sheep hair follicle stem cells, which were isolated and cul- tured, in order to establish the fine-wool sheep hair follicle stem cells culturing system in vitro. It separated and cultivated the fine wool sheep hair follicle stem cells by three methods, one was the two-step enzyme digestion, another one was mechanical separation, and the third was the combination of two-step enzyme digestion and mechanical separation. By measuring the num- ber, cloning efficiency and proliferation ability of hair follicle stem cells, the experiment intended to evaluate the advantages and disadvantages of the three methods synthetically, for not only obtaining a large number of hair follicle stem cells conveniently, but also seeking out ideal culture conditions of reducing their differentiation. Under the inverted microscope, the cells obtained by three cultural methods were paving stone-like, by which expressed keratin K19 and integrin-131, indicating successful ac- quirement of sheep hair follicle stem cells and establishment of in vitro culture system. Finally, the experiment showed that, it was the optimal method to obtain and culture hair follicle stem cells by the combination of two methods of two-step enzymatic digestion and mechanical separation.