| Background and objective:Hair loss disorders,such as androgenetic alopecia and alopecia areata,could affect patients'appearance,and also have a negative impact on their psychological burden,which seriously affects the quality of their life.With the development of the economy and the continuous improvement of people's living standards,people's expectations improve,as well.Further studies are needed to select drugs that effectively promote hair growth and hair follicle regeneration.Recently,the important role of fibroblast growth factors(FGFs)in development,diabetes,tumorigenesis and metastasis has been paid more attention.In the field of hair research,the FGF9 subfamily(FGF9,FGF16,FGF20)has been paid more attention in the development of hair and the regulation of hair biological behaviors.In our previous work,we have detected several growth factors'effects in regulating hair growth,including EGF,aFGF,bFGF and FGF10.We found that some growth factors could promote hair growth,especially FGF20 has potential role in regulating hair growth/regeneration.However,the exact mechanisms of FGF20 on hair need to be further studied.We intend to detect the effects of FGF20 on hair growth,cycle transition,and regeneration in hair follicle organ culture model,intradermal injection of FGF20animal model,and Patch Assay model.And we further explore whether Wnt singnaling pathway plays a role in the regulation of hair follicles after the application of FGF20.Material and Methods:1.Subjects:C57BL/6 mice,nude mice.2.Study on the regulation of FGF20 on the growth of hair follicles:We construct a mouse vibrissal follicle organ culture model.The mouse vibrissal follicles were divided into 4 groups:the group A was the control group,the group B was added 10ng/ml FGF20,the group C was added 100ng/ml FGF20,and the group D was added 1000ng/ml FGF20.(1)The length of each follicle was measured under the dissecting microscope on baseline and 1 week post-culture.(2)Collect the vibrissal follicles from control group and FGF20100ng/ml group 3 days after culture,and prepare paraffin embedded slides.HE staining was performed to observe the morphology of vibrissal follicles after culture.(3)Collect hair follicles from control group and FGF20 100ng/ml group 3 days after culture,prepare paraffinembeddedslides,anddetecttheexpressionlevelofKi67by immunofluorescence.3.Study on the regulation of FGF20 on the follicle cycle transition of mouse:Sixty female 60-day-old C57BL/6 mice were randomly divided into 2 groups,the control group and the FGF20 group.For the control group,250?l DPBS was intradermal injected into the back skin,once a day for 14 days.For the FGF20 group,250?l FGF20 solution(including 1?g FGF20)was intradermal injected into the back skin,once a day for 14days.(1)Observe the state of each mice every day,count the number of mice in the anagen phase,and take a picture.(2)Collect the dorsal skin of 5 mice from both groups,prepare the single cell suspension of epidermis and hair follicles,and perform flow cytometry.The flow cytometry detected the proportion and proliferation of CD34~+CD49f~+hair follicle stem cells and CD34~-CD49f~+Pcad~hii hair follicle precursor cells.(3)Collect the dorsal skin samples of 5 mice from both group 3 days,7 days,10days,and 14 days post-treatment.Collect the dorsal skin samples of other 5 mice from both group 10 days post-treatment.Perform HE staining of samples of 3 days,7 days,10days,and 14 days post-treatment.(4)Total RNA was extracted from 3 samples of both group at 10 days post-treatment and PCR Array of the Wnt signaling pathway was performed.(5)To confirm the differentially expressed genes selected by PCR Array(Wnt5a,Fzd4,Lrp5,beta-catenin,Lef1,and Tcf1),qPCR was performed with samples of 3 days,7 days,10 days,and 14 days post-treatment.(6)Immunohistochemistry and immunofluorescence staining of the differentially expressed genes selected by PCR Array(Wnt5a,Fzd4,Lrp5,beta-catenin,Lef1,and Tcf1)was performed with samples of3 days,7 days,10 days,and 14 days post-treatment.(7)Western Blot was performed to detect the differentially expressed genes selected by PCR Array(Wnt5a,Lef1,and Tcf1)with samples of 3 days,7 days,10 days,and 14 days post-treatment.4.Study on the regulation of FGF20 on hair follicle regeneration:The Patch Assay model was constructed with the newborn mouse epidermal cells and vibrissal follicle dermal papilla cells.They were divided into control group(epidermal cells+dermal papilla cells)and FGF20 group(epidermal cells+FGF20 100ng/ml cultured dermal papilla cells+FGF20 100ng/ml).(1)The samples of both groups were collected 14 days after the construction of Patch Assay.The size and the number of regenerated hairs of each Patch Assay was counted.(2)HE staining of the samples was performed and the structure of Patch Assay was observed under microscope.Results:1.FGF20 induced the growth of cultured vibrissal follicles:(1)In a certain range,the growth rate of the cultured vibrissal follicles increased with the increase of FGF20concentration,and the growth of the FGF20 100ng/ml group was significantly increased than that of the control group.However,too high FGF20 concentration(1000ng/ml)could inhibit the growth of the vibrissal follicles.(2)HE staining did not show much differences of the morphology of vibrissal follicles between control group and FGF20100ng/ml group.(3)Ki67 immunofluorescence staining showed that the number of Ki67~+matrix cells in the FGF20 100ng/ml treated group was significantly higher than that of the control group,indicating that the proliferation of the follicles in the FGF20treated group was significantly higher than that of the control group.2.FGF20 induced the telogen-anagen transition of mouse hair follicles:(1)The mice in the FGF20 treated group entered anagen phase since 6 days post-treatment,which was significantly earlier than that of the control group.Most FGF20 treated mice entered the anagen phase 8 days post-treatment,while only 40%of control group mice entered the anagen phase 14 days post-treatment.(2)HE staining showed that both groups were at telogen phase 3 days post-treatment.Some of the FGF20 treated mice entered the anagen phase,while the control group was still at the telogen phase 7 days post-treatment.Almost all mice entered the anagen phase in the FGF20 treated group 10 days post-treatment,while only small part of control group mice entered the anagen phase 10days post-treatment.Almost all mice entered middle or late anagen phase in the FGF20treated group 14 days post-treatment,while only small part of control group mice entered early anagen phase 14 days post-treatment.(3)Flow cytometry showed that the proportion and proliferation of CD34~+CD49f~+hair follicle stem cells in the FGF20treatment group was significantly higher than that of the control group.But there was no significant difference in the proportion and proliferation of the CD34~-CD49f~+Pcad~hii follicle precursor cells between the two groups.(4)PCR Array test identified that 27genes were more than 2.5 folds changed among the 84 Wnt signaling pathway related genes.The expression level of Wnt5a,Fzd4,Lrp5,Lef1,and Tcf1 was up-regulated in the FGF20 treated group.The expression level of the key factor of Wnt signaling pathway,?-catenin,was also significantly different(1.91 folds).(5)The RNA expression level of Wnt5a,?-catenin,and Lef1 in FGF20 treated group was higher than that of the control group since 3 days post-treatment.The RNA expression level of Wnt5a,Lrp5,Fzd4,?-catenin,Lef1,and Tcf1 in the FGF20 treated group was higher than that of the control group 7 days and 10 days post-treatment.The RNA expression level of Wnt5a,?-catenin,Lef1,and Tcf1 in the FGF20 treated group was still higher than that of the control group 14 days post-treatment.(6)Immunohistochemical staining/immunofluorescence staining showed that Wnt5a expression level was higher in the FGF20 treated group since 3 days post-treatment.There was no significant difference in the expression level of Lrp5 and Fzd4 between the control group and the FGF20 treated group.The expression level of?-catenin and Lef1 was significantly higher in the FGF20treated group than that of the control group since 3 days post-treatment.The expression level of Tcf1 in the FGF20 treated group was significantly higher than that of the control group 3 days post-treatment,however,there was no significant difference at other time points between the two groups.(7)Western Blot showed the expression level of Wnt5a and Lef1 was significantly increased in the FGF20 treated group than that of the control group since 3 days post-treatment.Although the expression level of Tcf1 in FGF20treated group was significantly higher than that of the control group 3 days post-treatment,however,there was no increase at other time points in the FGF20 treated group.3.FGF20 induced the regeneration of hair follicles:(1)We successfully constructed the Patch Assay model by injecting the mixture of newborn mouse epidermal cells and the cultured vibrissal follicle dermal papilla cells into the nude mice.(2)The size and number of regenerated hair follicles was increased in the FGF20 treated group.(3)HE staining showed that the structure of Patch Assay was more complex in the FGF20treated group than that of the control group.Conclusion:1.FGF20 can promote the growth of vibrissal follicles through upregulating the proliferation of hair matrix cells.2.FGF20 can promote the telogen-to-anagen transition of hair follicle in mice.FGF20 regulates the cycle of hair follicle by promoting the proliferation and differentiation of hair follicle stem cells.FGF20 up-regulated Wnt5a to activate the Wnt canonical signaling pathway and then regulate the cycle of hair follicles.3.Through the Patch Assay model,we successfully constructed the chimeric follicle of newborn mouse epidermal cells and mouse vibrissal follicle dermal papilla cells.FGF20 can induce the regeneration of hair follicles through Patch Assay.4.FGF20is a promising therapeutic agent to stimulate hair growth and regeneration.The exact mechanism,optimum concentration and treatment strategy should be further studied. |
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