The Research Of Identifying Rat Hair Follicle Stem Cell And Selecting The Optimal Culture Medium

Objective:To identify hair follicle stem cells and observe the effects of three different culture media on the proliferation of hair follicle stem cells.Method:The cleaning skin of the rat's beard were dissected under a stereocroscope and digested by Dispase? firstly and by the mixture of trypsin and EDTA secondly.Using ? collagen and differential adhesion methods obtain hair follicle stem cells.To observe cell morphology under an inverted microscope and identify by Giemsa staining, flow cytometry, CD34, ?1-integrin (CD29),CK15detection,alizarin red staining and oil red O.Cell suspension was divided into three pieces based on the average number of cells and respectively cultured by keratinocyte serum-free medium,DMEM/F-12+10%fetal bovine serum and keratinocyte serum-free medium+10%fetal bovine serum.To indirectly determine the impact of three different media for hair follicle stem cells generated by cell viability and growth curve and flow cytometry.Result:Rat hair follicle stem cells grew well,CK15,CD34,(31-integrin(CD29) expression was positive.After induced by osteogenic inductor,calcified nodules appeared after alizarin red staining.After oil red O staining, fat droplets were visible within the cytoplasm.There was no difference in cell survival rate among the three groups (P>0.05).Growth curves showed:the growth rate of keratincyte serum-free medium+10%fetal bovine serum> DMEM/F12+10%fetal bovine serum> keratinocyte serum-free medium (P<0.05).Flow cytometric examination showed the expressions of CD34,CK15and ?1-integrin (CD29) in the DMEM/F-12+10%FBS were lower than those in the other two groups (P<0.05).The expression of CD34in the keratinocyte serum-free medium+10%fetal bovine serum group was lower than that in the keratinocyte serum-free medium group (P<0.05),?1-integrin (CD29) and CK15was no statistically significant difference.Conclusion:The identification results show that using the above method for culturing rat hair follicle stem cells in good condition.The cells contain hair follicle stem cell surface markers and have differentiation ability.We can obtain higher purity hair follicle stem cells in the Keratinocyte serum-free medium than in the DMEM/F12+10%serum-free medium, serum-free medium in keratinocyte serum-free medium+10%serum-free medium can promote cell proliferation.