Study On The Expression And Purification Of FGF5 And Its Effect On 3D Cultured Dermal Papilla Spheroids

Objective(1)To construct rh FGF5 recombinant plasmids by prokaryotic system,express and purify it.(2)To construct 3D cultured dermal papilla model,and study the effect of rh FGF5 on dermal papilla and its correlation with androgenic alopecia.Methods(1)rh FGF5 was expressed by p ET3a-rh FGF5/E.coli BL21(DE3)p Lys S,and was purified by a CM Sepharose Fast Flow chromatography and Heparin Sepharose Fast Flow chromatography.(2)Identification of rh FGF5 was done by Western Blot,and the activity of rh FGF5 was detected by Western Blot using NIH/3T3 cells.(3)Dermal papilla cells(DPCs)and neonatal rat epidermal cells were extracted by enzyme digestion method;DPCs were identified by Toluidine blue staining,Alcian blue staining and ALP immunofluorescence.(4)Hair follicle dermal papilla spheroids were established by DPCs cultured in 3D by ultra-low adhesion method;Cell activities were detected by LIVE/DEAD kit;The functions of dermal papilla spheroids were tested by q PCR and WB methods.(5)The effect of rh FGF5 on dermal papilla spheroids and androgenic alopecia were detectd by WB.Results(1)rh FGF5 was successfully expressed using p ET3a-rh FGF5/E.coli BL21(DE3)p Lys S,and then purified by CM Sepharose Fast Flow and Heparin Sepharose Fast Flow;The identification of rh FGF5 was done by its antibody;The activity of rh FGF5(20 ng/ml)were tested in NIH/3T3 cells.(2)Enzyme digestion method obtained relatively pure DPCs and neonatal mouse epidermal cells.(3)DPS was established successfully,which was used to detect the promoting effect of minoxidil on DPS.(4)rh FGF5 up-regulated the expressions of BMP4 and Wnt5 a,and down-regulated the expression of ?-catenin in DPS,thereby inhibiting the hair induction ability of DPS;The expression of FGF5 in epidermal cells after the treatment of DHT was significantly up-regulated.