毛囊各细胞体外的不同分离、培养及其生物学特性

Isolation and cultivation of the cells of hair follicles in vitro,and observation of the biological characteristics of them.

目的进行毛囊各主要生长细胞的体外分离、培养及生物学特性的观察。方法对人头皮毛囊组织进行体外钝行分离、组织块法及酶消化来获得毛囊外根鞘细胞(ORSC)、毛乳头细胞(DPC)、毛囊真皮鞘细胞(DSC),利用不同的体外培养条件分别对这三种细胞进行体外增殖培养,利用倒置显微镜及免疫组化的方法对细胞来源和生长特性进行观察。结果组织块法培养的上、下段毛囊:上段毛囊来源的ORSC增殖生长速度较下段毛囊ORSC快;酶消化法分离得到的ORSC,利用K-SFM培养液进行培养,整体细胞生长呈典型的"铺路石"样结构,可传4代;酶消化法分离得到的DSC,利用10%～15%低糖培养液进行培养,单个细胞生长状态呈长三角形,可传三代;经酶消化法,每3 cm×3 cm大小的头皮可以获得约500个完整毛乳头,利用10%～15%低糖培养液进行DPC培养,细胞由毛乳头边缘向四周呈放射状生长,成功传代8代,四代内的细胞"聚集性"生长状态明显。免疫组化细胞鉴定:ORSC CK19免疫组化染色呈阳性;DPC浆抗波形丝蛋白呈阳性显色;DSC胞漿内高表达波形丝蛋白。结论组织块法证明毛囊上段细胞生长情况更为活跃,再次验证毛囊干细胞可能存在于毛囊上段"隆突部"的假说;经钝行分离和酶消化法结合的方法来进行人头皮毛囊相关细胞的分离和培养,所获得三种细胞的细胞活性、生长增殖情况均较好,细胞鉴定证实细胞来源的可靠性。

Objective To isolate and cultivate the major growth cells of hair follicles in vitro, and observe the biological characteristics of them. Methods Application Blunt line separation in vitro. Tissue crawling method and Enzymatic digestion method were used to obtain ORSC ,DPC,DSC, and cultivating the above cells by different culture conditions in vitro. Inverted microscope and immunohistochemical methods were used to observate the source of cells and the growth characteristics of them. Results Cultivation of the upper and under segments of hair follicle was used by Tissue crawling method ： ORSC from the upper hair follicle had faster growth rate than the under ; Application of Enzymatic digestion method to get ORSC and use K-SFM culture medium to culture cells. The overall ceils growth as typical ＂cobblestone＂-like structure can be subcultured for 4 generations. Application of Enzymatic digestion method to get DSC and use 10% 15% low -Sugar culture medium to cultivate cells, single cell grow like Long- triangle can be subcuhured for 3 generations, After Enzy- matic digestion method, each size of 3× 3cm^2 scalp can get about 500 dermal papilla tissues, the usage of 10% - 15% low-Sugar culture medium to cultivate DPC, DPC growing Radiation-like from the edge of the dermal papilla can be subcultured for 8 generations, at the same time, ＂clustering＂ growth clearly can be seen within four generations of cells. Immunohistochemical identification of ceils ： ORSC CK19 immunohistochemicat staining positive; DPC plasma anti-vimentin-positive color; DSC high cytoplasmic expression of vimentin. Conclusions By Tissue crawling method,it was proved that cells from the upper hair follicle had more active growth. Validating the hypothesis that the hair follicle stem cells may be in the upper hair follicle ＂ bulge＂ again. Application of the blunt line separation and Enzyme digestion method to get hair follicle relative cells from human scalp Those three types of cells all are obtained better cell activity, growth and proliferation, cell identification has confirmed the reliability of the source.