| The research was conducted to find the optimal mink hair follicle culture conditions and establish the culture model in vitro. We also studied different methionine level on hair follicle growth, dermal papilla cells(DPCs) cycle and apoptosis, and some gene expressions of hair follicle, to confirm the influence rule of methionine on hair follicle of mink, which may lay the foundation of exploring molecular mechanisms of growth factors which influence hair follicles and improving the quality of animal furs.Hair follicles of mink were isolated by collagenase digestion and micros-dissection and cultured in Williams E serum-free medium, Williams E + 10% serum, DMEM serum-free medium, DMEM + 10% serum to select the optimal medium in sterile conditions; After that took the hair follicles in the optimal medium were cultured under the conditions of 29℃,31℃,33℃,35℃,37℃ and 39℃, respectively, to find the optimal suitable temperature. Hair follicles of mink were cultured in plates containing 500 μL of Williams E medium, and supplemented with 0, 0.01, 0.1, 1, 2, 4, 8, 16, 32 and 64μg/mL methionine, measured the length observed morphological changes of hair follicle for 8 days. Depending on results of experience, as the representative concentrations,hair follicles were cultured in plates containing 500 μL of Williams E medium supplemented with 0μg/mL(control group), 8μg/mL(promoted the growth of mink hair follicles) and 64μg/mL(inhibited hair follicles growth).Then total RNA of hair follicle was extracted, after 48 hours and the expression of IGF-Ⅰ and EGF were detected to analyze the effects of different concentrations of methionine on gene expression by RT-PCR. The two steps method was utilized to isolate DPCs and cultivated cells were identified by immunofluorescence and immunohistochemical staining for α-SMA. DPCs were cultured in plates containing 2mL of DMEM medium+10%FBS supplemented with 0, 2, 4, 8, 16, 32 and 64 μg/mL methionine of the fourth passage cells, the cell proliferation was detected by MTT assay, and flow cytometry was used to evaluate the effects of Met on cell cycles and apoptosis of DPCs.The results showed that hair follicles cultured in vitro grew fastest in serum-free Williams E medium(P<0.05), hair follicles growth rate significantly decreased with the increase of cultured time and the optimum temperature was 31℃ which improved the follicles growth better than others(P<0.05). Adding less than 32 μg/mL methionine showed significant length growth of hair follicles compared to control group, especially in 8 μg/mL group(P<0.05), and the shortest one was in 64 μg/mL group, which was significantly different from than control(P<0.05); The highest expression of IGF-Ⅰin 8μg/mL group and the highest EGF gene expression was in 64μg/mL group. Compared with the control group, methionine at the concentrations of 2, 4, 8μg/mL could significantly promote the DPCs proliferation(P<0.05), especially in 8μg/ mL group, but it suppressed the proliferation of DPCs at the concentration of 64μg/mL. Cells in G1 phase of 8μg/mL methionine group was less than control group, and cells proliferation index, S and G2 phase were significantly higher than other groups, the concentration of methionine did not affect the mink DPCs apoptosis(P>0.05).In conclusion, the optimal medium for mink hair follicles in vitro was serum-free Williams E, with the suitable condition 31℃, and certain humidity, 5% CO2. The study suggests the addition of 8 μg/mL methionine caused the highest relative expression of IGF- Ⅰ,thus promoting hair growth obviously. Adding appropriate concentration of methionine in the culture medium can promote the proliferation of DPC, enhance cell proliferation activity. |
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