Optimization Of Dermal Papilla Cell Cmltivation And Effects Of MiR-206 On Its Founction In Cashmere Goat (Capra)

Dermal papilla cells (DPCs) play key roles in the cyclic regulation of hair follicles (HF) by inducing the regeneration of HF and controlling the size of HF, to some extent. This study aimed to optimize the cultivation system of DPCs derived from cashmere goat (Capra hircus) skins. Mean time, microRNA-206 (miR-206) was transfected into DPC and the mRNA expression of 7 cycling-related genes were investigated, to explore its effects on hair follicle cycling in cashmere goats. Skin samples at anagen period were collected and separated by combination of blunt dissection, neutral protease and collagenase Ⅱ to obtain single hair follicle and release dermal papilla (DP). After that five sets of cell culture medium were formulated, and immunofluorescence staining method was used to detect the expression of a-smooth muscle actin (a-SMA) and CD 133 in DPCs; After transfecting miR-206 mediated by Lipofectamine(?)3000, the mRNA expressions of 7 genes related to hair follicle cycling were measured by qRT-PCR. Results showed that:DPCs were successfully cultivated within 3 days for attachment as well as for amplification. The efficacy of attachment and amplification time by adding (3-Estradiol (β2), insulin-transferrin-selenium (ITS), and epidermal growth factor (ECF) individually was higher than that in the control group, while the efficacy of these 3 factors is:ITS>EGF>β2; Compared with other groups, the medium with β-Estradiol ((32), insulin-transferrin-selenium (ITS), epidermal growth factor (EGF) had better attachment and amplification time, as well as cell morphology and agglutination. We suggested that the culture components for secondary hair follicle DPC (SHF-DPCs) at anagen period were Dulbecco’s Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F12)(1:1),10% Fetal bovine serum (FBS), double resistance (penicillin and streptomycin,100 IU), β2 (0.5 μg/mL), ITS (3 μg/mL), EGF (10 μg/mL). Compared with the control group, transfecting miR-206 increased the expression of Protein Tyrosine Phosphatase, Non-Receptor Type 1 (PTPN1) and Bone morphogenetic protein 2 (BMP2) (p<0.01), while decreased the expression of BCL-2 assoceated athanogene 4 (BAG4), MAX dimerization protein 4 (MXD4), insulin like growth factor 1 (IGF-1) and fibroblast growth factor receptor 2 (FGFR2) (p<0.01), and had no effect on BMP4 expression (p>0.05). We therefore assumed that BAG4 maybe the candidate target gene of miR-206. In conclusion, we optimized the cultivation system of DPCs in cashmere goats, and miR-206 was stably overexpressed in SHF-DPCs, which affected the expression of several cycling-related genes in hair follicles, and thereby might inhibit the development of hair follicle at the anagen period. Our results will facilitate the exploration of molecular mechanism of miR-206 in the cyclic regulation of HF.