Effects Of VEGF And L-methionine On Proliferation And Differentiation Of Secondary Hair Follicle Outer Root Sheath Cells Of Cashmere Goat In Vitiro

This study was conducted to isolate and culture goat skin primary and secondary hair follicle outer root sheath cells(pHFORSC and sHFORSCs) in vitro, and evaluate the effect of vascular endothelial growth factor(VEGF) and L-methionine on the viability, proliferation, differentiation of sHFORSCs as well as the FGF5 mRNA expression level in cells. The object is to explain the mechanism of the outer root sheath cells in hair cycle of secondary hair follicle, and to provide theoretical support for further study of development mechanism of secondary hair follicle of cashmere goat.Skin collection of Shanbei white cashmere goat, through cut into pieces,digestion, stripping isolated the primary and secondary hair follicle outer root sheath cells, then digest hair follicle to obtain outer root sheath cells, and the cells was cultured under 37 ℃in a 5%(v/v) CO2 atmosphere and 100% humidity. MTT and EdU were adopted to determine the effect of dose and time of VEGF and L-methionine on the viability and proliferation of s HFORSCs. Primers were designed based on the sequences of PCNA, K10 and FGF5 cDNA in Carpra which were deposited in the GenBank Data Library. Then, mRNA expression level of PCNA, K10 and FGF5 after treatment was weighted by real-time PCR.The main results showed that as follows:1. pHFORSC and s HFORSCs were successfully isolated and cultured in vitro. pHFORSCs showed positive immunoactivity of CK17, and sHFORSCs showed positive immunoactivity of CK17 and CK15, which are main biomarkers of ORSCs. pHFORSC and s HFORSCs have the growth curves for the "S" shape, there are some differences in shape and size between pHFORSCs and sHFORSCs.2. Addition of VEGF in the cell culture medium can promote the proliferation of s HFORSCs, inhibits their differentiation and inhibit the expression of FGF5.When treatment with VEGF of 100 ng/mL, the cell vibility(48 h), proliferation cells number and expression of PCNA mRNA were the highest, and significantly higher than the control group(P<0.01), cell viability(24 h) was significantly higher than control group(P<0.05). The cell activity and expression of PCNA mRNA at 24 h were higher than 48 h. With increasing concentration, the inhibitory effect of VEGF on cell differentiation is increasing, K10 mRNA expression of minimum at 100 ng / ml; With increasing time, tthe inhibition of VEGF on cell differentiation is enhanced, 100 ng / mL at 48 h was significantly lower than 24 h(P <0.05). With increasing concentration, FGF5 mRNA expression level of the lowest at 100 ng / mL group, and speculated that VEGF can inhibit the expression of FGF5 promote hair follicle growth.3. Addition of L-methionine in the cell culture medium can inhibit the proliferation and differentiation of sHFORSCs. When treatment for 24 h, with the increasing concentration, the inhibition of L-methionine was increasing, when 120 μg/mL strongest inhibition, but no significant; When treatment for 24 h, with the increasing concentration, the inhibition of L-methionine was first rise after falling, the 100 μg/mL inhibition of the most significant, very significantly higher(P <0.01), while 80 μg/mL PCNA mRNA expression level of the minimum, no significant difference; the cell viability of sHFORSCs at 48 h in each group were less than 24 h. When treatment for 24 h, with increasing concentration, tExpression level of K10 mRNA also showed a downward trend, 120 μg/mL group was the lowest, no significant difference; when treatment for 48 h, the expression of K10 mRNA decreased after the first while rising, 80 μg/mL group was the lowest, no significant difference; the high concentration of L- methionine(120 μg/m) to inhibit the effect of sHFORSCs proliferation and differentiation are weakened.Results indicate: First time successful culture pHFORSC and sHFORSCs of cashmere goat in vitro; VEGF promotes the proliferation of s HFORSCs, exert a role in inhibition of cells differentiation, and might participate the cycling of hair follicle by suppression of FGF5; L-methionine can inhibit the proliferation and differentiation of sHFORSCs.