Junctional Adhesion Molecular-1Functions In Promoting Hair Growth

BackgroundMost of the adult mammalians furred themselves by hair. This provided an effective wayto protecting the skin, warming the body and signaling social connections and position.Hair follicles undergo cyclical bouts of growth (anagen), destruction (catagen) and rest(telogen). Epithelial stem cells in the bulge of hair follicle undergo a change of migration,proliferation and differentiation throughout the hair cycle. Epithelial stem cells support hairgrowth in tissue homeostasis and provide cell resources for wound healing. Unbalanced ofself-renewal and differentiation of epithelial stem cells happened in some Pathologyconditions, thus cause some diseases such as hair loss.Nude mouse can be used as an animal model for Xenograft owing to the deficits inimmune system. Another phenotype in nude mouse is hairless. So it can be involved instudies of hair growth. The deficits in immune system due to defects in thymus arerevealed in the first two years after the nude mouse discovered. Following studiesaccounted that the defects in epithelial cell development can be due to deficit in the gene ofFoxn1. Nude mouse can be involved in the studies of hair follicle reconstruction owing tothe deficits in immune system. The phenotype of hairless and deficits in immune systemare independent as engraftment of thymus do not lead to hair growth. However, those twoindependent phenotypes can be associated with the defects of Foxn1. Transcriptional factorFoxn1functions as a molecular switch regulating proliferation and differentiation ofepithelial cells. Epithelial cells with defects in Foxn1exhibit enhanced proliferation andreduced terminal differentiation. Targets of Foxn1include some genes relative topromoting differentiation of karatinocytes.JAM1can be localized at tight junctions of epithelia and endothelial cells. It is a typeⅠ transmembrane protein with a transmembrane domain, an extracellular domaincontaining two Ig-like loops. The two Ig-like loops in extracellular domain refer to D1loop and D2loop. JAM1can be dimerization through the extracellular domain. Cytoplasmtail of JAM1containing PDZ binding domain can be involved in the interaction of PDZ domain proteins such as ZO-1, PAR-3and AF-6. Those proteins are associated with tightjunction formation, cell polarity and cell migration.In our previous studies, hMSC（human early embryo mesenchymal stem cells）promotehairregenesis in nude mouse. This process accompanied by a change in expression ofpolarity proteins of JAM1, PAR-3and TIAM-1. Interfering expression of JAM1byconstruct of shRNA, hMSC lose the ability of promoting hairregenesis in nude mouse.This correlates the idea that JAM1has an essential role in promoting hairregenesis. Thedetailed functions of JAM1in this process need to be studied. In this thesis paper, weinvestigate the function of JAM1in hMSC cells and promoting hairregenesis in nudemouse.Part1: establishment of JAM1over-expression system.Methods: Plasmid construction using vector of eGFP-N1and coding sequence ofJAM1which gained by PCR using template of cDNA from HaCat cells. JAM1and eGFPwere constructed into fusion protein. A District stripe single bright of JAM1codingsequence can be gained. DNA fragments obtained connected with the digested vector, andthen transducted into E. coli to grow colonies with the antibiotic resistance. Plasmidamplified by transducted into E. coli cloning sequenced to identify an exact matchconstruct. Transfected to HEK-293T cells by the liposome, and observed fluorescenceexpression and localization. The successfully identified plasmid of the JAM1andinterference plasmid sent to the company to get the lentivirus.Results: The recombinant vector was transformed into DH5α. Positive clones wereselectes by PCR tests and sent to sequencing, the results of an exact match with the CDSsequence. To elaluate the expression of construct in cells, plasmid was get from activatedbacteria. JAM1expression in HEK-293T cells is mainly concentrated in the cell-celljunctions, punctate and zonal distribution. This indicates that correct reading frame fusionprotein was constructed successfully. The infection efficiency of lentivirus can reach90%.Part2: the impact of JAM1on cell migration and contact inhibitionMethods: based on the technology platform established, primary culture to get hMSCcells. JAM1and interference and its corresponding control virus infection of hMSC Hacatcells and get stable transfection of cell lines. Transwell migration assay been used toevaluate the expression and impact of JAM1on cell migration. In the case of similar celldensity, immunofluorescence detected of the YAP protein levels and subcellularlocalization in order to measure the occurrence of contact inhibition. Results: In this study, a stable growth of hMSC cell lines was identified with stem cellproperties, and showed a typical mesenchymal-like growth. Successfully get cells ofhMSC, Hacat the JAM1over expression and interference and control of stable transfectedcell lines. Transwell migration assay shows JAM1overexpression group have more cells tothe contralateral side after six hours migration, indicating that the JAM1molecules canpromote cell migration. The JAM1affect the degree of contact inhibition in epithelial cells.The group of JAM1overexpression is prone to contact inhibition. YAP is more susceptiblelocated in the cytoplasm.Part3: the effects of JAM1in hMSC cell migration and differentiationMethods: JAM1and interference, and its corresponding control virus were infectedwith the hMSC stable transfection CM-Dil dye labeled cells, intradermal injection totransplant into nude mice. Hair regeneration in nude mouse been observed. Paraffinsections and H&E staining techniques, based on morphology to determine the progress ofthe hair follicle cycle. The distribution of labeled cell in skin can be detected byimmunohistochemistry and immunofluorescence after paraffin sections and frozen sections.The expression of polarity molecular also been detected.Results: cells can be CM-Dil labeled highly efficiency. Red fluorescence is mainlydistributed in the cell membrane structure, including the structure of vesicles in the cellmembrane and cytoplasm. Frozen sections observed under fluorescence microscope, thecells can be successfully transplanted into nude mice skin, local positioning intradermal.JAM1over-expression group widely distributed with enhanced ability in vivo migrationability, located in the hair follicle and can be distributed in the outer root sheath. H&Estaining indicate JAM1over-expression group have more hair follicles in anagen, and invitro observation of hairregenesis is better than other groups. The JAM1over-expressiongroup in the hair follicle structure also showed co-localization of the epithelial tissuemarker K19, indicating that the hMSC effectively differentiated into epithelial cells,polarity molecules also been expressed in the JAM1overexpression group of hair follicles.CONCLUSION: The early embryo mesenchymal cells in nude mouse skin caneffectively promote the regeneration of hair, polar molecules JAM1play an important rolein this process, it promote cell migration in vitro and in vivo as well as the cells toparticipate in the hair follicle structures and contribute to cell differentiation. JAM1is agood candidate molecule in hMSC to manipulate to promote hair regeneration in nudemice.