Isolation And Culture Of Hair Follicle Stem Cells From Yangtze River Delta White Goats And Effects Of CMTM3 Gene On The Proliferation Of Hair Follicle Stem Cells

Hair follicle stem cells(HFSCs)belong to adult stem cells,which are the primitive cells in hair follicle(HF)and have the potential of multidirectional differentiation.Hair follicle stem cells are located in the hair follicle bulge,which can produce the cells required by the body through differentiation and proliferation,and are closely related to the maintenance of hair cycle,tissue renewal,and skin injury repair.Hair follicle stem cells are closely related to the hair follicle cycle,including growth phase,degeneration and rest periods.Hair follicle cycle is divided into three stages according to the activity of hair follicle stem cells and hair follicle morphology:growth stage,degeneration stage and resting stage.At present,the methods of separating hair follicle stem cells mainly include mechanical separation and enzymatic digestion.This study compared the methods of hair follicle stem cell isolation and purification published at home and abroad.Two methods for the isolation and culture of hair follicle stem cells from newborn Yangtze River Delta White Goats in vitro were found through comparative experiments,named "Experimental Method" and "Control Method".The best method was found by comparing the cell morphology,cell viability,resuscitation survival rate,cloning formation rate and expression level of hair follicle stem cell markers isolated and cultured by the two methods in vitro.This study also constructed CMTM3 overexpression and interfering adenovirus vector to explore its biological function in the proliferation process of hair follicle stem cells,we also investigated the effect of physiological concentrations of androgens on the proliferation of hair follicle stem cells.This study shows that:(1).The primary hair follicle stem cells isolated and cultured by Experimental Method grew faster and showed good proliferation and division,and presented typical paving stone shape.The positive rate of hair follicle stem cell markers reached more than 90%.The hair follicle stem cells isolated and cultured by Control Method are inferior to the Experimental Method in cell vitality,positive rate of hair follicle stem cell markers and other aspects,but this method is simple and convenient to operate.(2).The expression levels of DUSP6,DUSP1,MAP3K1 and HSPA1 were significantly increased after overexpression of CMTM3 in hair follicle stem cells(P<0.05).The expression levels of DUSP6,DUSP1,MAP3K1 and HASP were significantly higher after transfection of adenoviral vector and addition of physiological concentrations of androgen(P<0.05).(3).The results of WB assay showed that the expression levels of MAP3K1,FOS and HSPA1 were significantly increased after transfection of over-expressed adenoviral vector(P<0.01).The expression of MAP3K1 was significantly increased after the addition of physiological concentration of androgen in the medium,and the expression level of FOS was significantly decreased(P<0.05).Expression levels of DUSP6 and FOS were significantly decreased after transfection with interfering vectors(P<0.05).(4).Increasing the expression level of CMTM3 in hair follicle stem cells can decrease cell viability,and the proportion of cells in G1 phase is significantly increased(P<0.05),and the apoptosis rate of hair follicle stem cells is significantly increased(P<0.05).After transfection of the interference vector,cell viability increased,and the proportion of cells in the S and G2 phases increased significantly.The addition of physiological concentrations of androgens can also increase cell viability and promote cell proliferation.It is suggested that inhibition of the expression of CMTM3 in hair follicle stem cells and the addition of physiological concentrations of androgen can promote the proliferation of hair follicle stem cells,and the roles of the two are synergistic.(5).The levels of androgen receptor(AR)and epidermal growth factor(EGF)were the highest in the high-quality brush hair(P<0.05),and lowest in the castrated ram.VEGFA and IGF-1 were the highest in the castrated ram.It is speculated that high levels of EGF are conducive to the production of high-quality brush hair in the Yangtze River Delta white goats.High levels of VEGFA and IGF-1 may promote the production of ordinary coarse hair by non-high-quality brush hair and castrated ram.To sum up,this study established two methods for the isolation of hair follicle stem cells from newborn Yangtze River Delta White Goats.The hair follicle stem cells isolated and cultured in vitro by Experimental Method showed higher activity and the highest expression of hair follicle stem cell markers.Over-expression or interference of CMTM3 gene expression level in hair follicle stem cells can affect the expression level of related genes in hair follicle stem cells.Increasing the expression level of CMTM3 gene in hair follicle stem cells can inhibit the proliferation of hair follicle stem cells and promote cell apoptosis.Inhibition of CMTM3 gene expression level in hair follicle stem cells can promote the proliferation of hair follicle stem cells,increase the proportion of G2,S phase cells,and inhibit cell apoptosis.Adding androgen of physiological concentration in the culture medium alone can also promote the proliferation of hair follicle stem cells.Inhibiting the expression of CMTM3 gene in hair follicle stem cells and adding androgen of physiological concentration had the most significant effect on promoting the growth of hair follicle stem cells.