| The regeneration and size of hair follicle(HF)is controlled by dermal papilla(DP),which is considered to be the control center of growth and cycle of HF,that makes it a good model for studying the mechanism of HF morphogenesis and development.However,the long time and low efficiency of dermal papilla cell adhesion and migration in vitro affect the experimental efficiency,so that it is limited as the experimental material to study the mechanism of hair follicle cycle.The aim of this experiment was to explore the optimal culture medium and the optimal proportion of serum for the culture of dermal papilla cells in order to improve their culture efficiency.The results indicated:(1)Isolation and culture of DPCs from hair follicle is laborious and time-consuming job,thus present study was designed to optimize the culture condition of goat DPCs in order to acquire enough DPCs for other studies.In this study,we separated and cultured DP from goat hair follicle under stereoscope by microdissection,and confirmed the identity of cultured cell by using antibodies specific to their marker genes CD133/prominin-1,?-smooth muscle actin,alkaline phosphatase(AKP)and Versican,then cell counting,alkaline phosphatas analysis,(3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide assay and other methods were used to compare the combination of serum concentration and medium types.DPCs shared the expression of specific markers as previously reported,and they were successfully cultured in four tested culture medium supplemented with different proportions of serum respectively;(2)Growth of goat DPCs depends on the nutritional supplement: the higher proportion of serum added in culture medium resulted faster proliferation rate and stronger ability to migrate and induce hair growth.Advanced medium does contain effective factors to maintain or strengthen DPCs proliferation and inducing ability,thus it is more suitable for propagating DPCs than conventional medium,otherwise when the serum addition proportion is 10%,the growth and proliferation of DPCs cultured advanced medium are significantly higher than that in conventional media(P<0.05).The effect of Advanced media added with 6% serum for DPCs propagation is as good as the normal medium added with 10% serum(P > 0.05).6-bromoindirubin-3-oximebio(BIO)is an inhibitor of glycogen synthase kinase(GSK-3?),can activate the Wnt signaling pathway,but whether BIO enhances the induction capacity of dermal papilla induction through bio remains to be explored.The results were as follows:(1)The dermal papilla cells were cultured in vitro on the medium supplemented with different concentration gradient BIO.The results showed that the higher the concentration of BIO was,the lower the proliferation ability of the cells was by using the method of CCK8 cell counting kit-8 colorimetry.The results of AKP assay showed that the induction capacity of dermal papilla cells was significantly increased with the addition of 1.5 ?M of BIO.(2)DPCs cultured without BIO were used as control group while ?-actin was used as internal reference gene.Real-time fluorescence quantitative PCR and Western blotting were used to verify the results: the expression of insulin-like growth factor-1 gene(IGF-1),fibroblast growth factor factor 7(FGF7),AKP,?-catenin was significantly up-regulated(P < 0.05)in cultured dermal papilla cells cultured with 1.5 ?M bio,while the expression of bone morphogenetic protein4(BMP4)was significantly down-regulated(P < 0.05).In this study,the optimal culture medium and serum concentration of hair papilla cells of cashmere goat in vitro were explored for the first time,and the important role of 1.5 ?M BIO in maintaining the induction activity of DPCs in hair follicles in vitro was explored.It provides an ideal cell model for further studying the molecular mechanism of DP regulating HF cycle development. |
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