Localization Of Skin Stem Cells Of Inner Mongolia Cashmere Goats

5-bromodeoxyuridine(BrdU), which was injected intravenously into the goats'neck, was used to label DNA of skin of Inner Mongolia Cashmere Goats. Experimental materials were took from back of the goats every week, then fixed in 4% paraformaldehyde, embedded with paraffin and detected by monoclonal antibody of anti-BrdU. The purpose of this study is to investigate the feasibility of labeling skin of Inner Mongolia Cashmere Goats with BrdU, and to optimize its immunohistochemical conditions. Except for this, we estimated location, migration track of skin stem cells according to a trait that BrdU marker has been in skin stem cells for a long time due to slow cycling of stem cells. The results showed that:Positive cells with red nuclear were observed in labeled goats. However, positive signals were not observed in the samples from unlabeled goats and those incubated with PBS instead of BrdU–antibodies from labeled goats. The optimal immunohistochemical results were shown in the conditions that denaturing DNA in 2M HCl for 30min at 37℃, and repairing antigens in trypsin for 30min at 37℃and the dilution of biotin-conjugated second antibody and streptavidin-peroxidase is 1:100.BrdU can be detected only 10 weeks in Inner-Mongolia goats and can be used for large animals in vivo short-term markers. The positive signal, nuclear labeled by BrdU, was present in the primary follicles, secondary follicles and epidermis, especially in the hair bulb and the outer root sheath. With labling time increasing, the trend of gradual migration of labeled cells were observed.The skin sample of Inner Mongolia Cashmere goat labeled BrdU was incubated in 0.25% protease at 4℃3h, then separated the hair bulb and outer root sheath with mechanical separation method. Adherent cultivating with collagen type IV for 10 min. 10 min later, the unattached cells were removed and the rapidly adherent epidermal cells were cultured in stem cell growth medium. Follicle stem cells were isolated from the region over the hair bulbs of primary follicles. The size of follicle stem cells were round or polygonal, with big and obvious nucleus, smaller organelles. P63 were positive in the outer root sheath and the hair bulb of hair follicle. These results idicated that there were skin stem cells in the population of the BrdU labeled cells.According to the trait that slow cycling, high proliferative capacity, adhesion ability and strong clonogenicity of stem cells got from the hair bulb and the outer root sheath, BrdU labeled cells were initially identified as a type of skin stem cells. The analogs of skin stem cells located in Inner Mongolia cashmere goat primary follicles, secondary follicles and epidermal tissue, especially in the hair bulb and the outer root sheath.