Experimental Investigation Of The Induction Of Epidermal Cells To Dedifferentiate With PEGFP-C3-bFGF

Objective 1.To construct a recombinant plasmid carrying human basic fibroblast growth factor(bFGF).2.To investigate whether human epidermal cells could be transfected with the eukaryotic expressive vector of pEGFP - C3 - bFGF and express the exogenous gene.Meanwhile to investigate the dedifferentiation of epidermal cells into their progenitor stem cells induced by pEGFP - C3 - bFGF,for exploring the possible signaling pathway involved in the reversion process of epidermal cells.3.To investigate the expression and distribution characteristics of fibroblast growth factor receptor 2(FGFR2) in the human fetal skin during various stages of the gestation,and to explore its biological significance in the cutaneous development and wound healing process.Methods 1.All experimental methods were performed according to the standard protocols of molecular clone technology.The bFGF cDNA was amplified by polymerase chain reaction(PCR) from PBR322 - bFGF with Hindâ…¢and EcoRâ… at both sides,and it was subsequently subcloned into the multiple cloning site of pEGFP - C3 to obtain the recombinant plasmid pEGFP - C3 - bFGF,followed by the identification of DNA sequencing analysis.2.pEGFP - C3 - bFGF was transferred into the human epidermal cells by using liposome - mediated method.Subsequently,the phenotypic changes of epidermal cells were detected by using immunohistochemical staining,immunofluorescent analysis,RT -PCR, western - blot 36 hours later.For controls,epidermal cells with no intervention treatment were cultured simultaneously.3.The expression of FGFR2 in the human fetal skin during various stages of the gestation was analyzed by using immunohistochemical method.Results 1.DNA sequencing analysis indicated that the recombinant plasmid pEGFP - C3 - bFGF possessed the same open reading frame as pEGFP and perfect potential to translate and express in epidermal cells.2.Specific green fluorescence can be observed by fluorescence microscope in the gene positive groups.Immunohistochemieal staining revealed that the expression levels of CK19 and CK14 were up- regulated,while those of CK10 significantly down- regulated. Meanwhile,immunofluorescent analysis indicated thatβ₁ integrin was expressed on the mem- brane of positive cells in lower level,CK19 and CK14 were expressed on the membrane and in the cytoplasm of those cells,CK10 was negative.Additionally,the expression levels of some putative biological markers in human epidermal stem cells were analysed by RT- PCR detection after pEGFP - C3 - bFGF treatment.The relative gene expressions of CK19 and CK14 increased in pEGFP - C3 - bFGF treatment group,whereas the expression of CK10 was observed to be significantly suppressed when compared to controls.Western -blot also proved the dedifferentiated phenomenon of epidermal cells in our experimental system.3.Positive immunohistochemical signals of FGFR2 were found in fetal skins,which were closely related to the developmental patterns of hair follicles.The expression of FGFR2 was first detected in the cellular membrane of epidermal cells in the undifferentiated epidermis during the embryonic period. Coinciding with the period of the stratification of the epidermis,FGFR2 was observed in the newly formed epidermal layers and primary hair germs.During the period of follicular keratinization, the differentiated spinous and granular layers were very prominent,and the epidermis reached to its maximal thickness at this time,meanwhile,the follicular buds also progressed through a rapid morphogenesis.The appearance of FGFR2 was observed exactly in the epithelial -mesenchymal interaction of hair bulbs at high level.Furthermore,during the period of follicular keratinization,the expression of FGFR2 was detectable in the epidermal layers,capillary endothelium,ducts of sebaceous glands,as well as the epithelial -mesenchymal interactions of follicles.However,the differential expression was coincided with the maturation and differentiation process of hair follicles at the end of this period.Conclusion 1.bFGF is cloned into the C terminus of pEGFP - C3.Under the control of enhancer and promoter of pEGFP- C3,expression of bFGF can be up- regulated without influencing the structure and function of its target protein.2.The pEGFP - C3 - bFGF is successfully transferred into human epidermal cells,which can induce epidermal cells to reverse their differentiated process and thus express some undifferentiated proteins of epidermal stem cells.3.The differential expression of FGFR2 in human fetal skins is closely related to the developmental patterns of hair follicles,which contributes to the maintenance and proliferation of follicle precursors,and regulates them to differentiate into terminal functional cells in the hair follicles.