A Comparative Study On Cellular Function Of Human GJB6 Gene Mutants

Background Hidrotic ectodermal dysplasia(HED,scilicet Clouston syndrome)is known as a rare autosomal dominant disorder.It is characterized by the triple sign of alopecia,nail dystrophy and palmoplantar hyperkeratosis.Hidrotic ectodermal dysplasia is caused by mutations in the human gap junction beta 6 gene which is used to encode connexin30(Cx30).So far,five missense mutations have been found,including G11R,A88V,V37E,D50N and N14S.At present,there are few studies on the pathogenesis of hidrotic ectodermal dysplasia.It is essential to conduct more in-depth study on the pathogenic gene(gap junction beta 6)in order to make more and further progress.In the early period,our research group successfully constructed stably expressed wild type and mutant type(A88V)of gap junction beta 6 gene in the HaCaT cell strains by Tet-on gene expression system at first,then they moved forward a single step by using the successfully constructed HaCaT cell strain with the strongest function mutation(G11R)to make the Affymetrix expression profile chip,to study the possible signal pathway and mechanism of gap junction beta 6,and through cytofunctional experiments further understood the mechanism of apoptosis of HaCaT cell caused by gap junction beta 6 mutation.The possible mechanism of different clinical phenotypes of hidrotic ectodermal dysplasia caused by gap junction beta 6 gene.In this study,HaCaT cell strains that stably express gap junction beta 6 gene and its three mutants(D50N?V37E?A88V)are continuously established by using Tet-on lentivirus as vector,and the cellular function comparison among different mutants is made through four kinds of cytofenctional experiment.Objective Four kinds of cellular functional experiments are carried out on HaCaT cell trains of gap junction beta 6 gene wild type and its three mutant types(D50N?V37E?A88V)which were successfully constructed by Tet-on gene expression system,besides the effects of different mutant types on the proliferation,apoptosis and senescence of cells are compared in groups,so as to explore the possible mechanism of gap junction beta 6 gene mutant types causing different clinical phenotypes of hidrotic ectodermal dysplasia in a more in-depth way and provides a functional researth basis for the subsequent typing study of hidrotic ectodermal dysplasia.Methods In this experiment,we used the successfully constructed wild type and mutant type(D50N,V37E,A88V)cell strains of gap junction beta 6 gene.Through MTT method and Brdu method to detect the effects of wild type and mutant type expression induced by DOX on the proliferation of HaCaT cells;through Caspase3/7 method to detect the effects of wild type and mutant type expression induced by DOX on the apoptosis of HaCaT cells;through Senescence-associated beta-galactosidase dyeing method to detect the effects of wild type and mutant type expression induced by DOX on the senescense of HaCaT cells.Results The result of MTT method demonstrates that the group of D50N+DOX has the highest inhibition of cell proliferation,followed by the group of A88V+DOX,and the group of V37E+DOX has the lowest inhibition of cell proliferation.The result of Brdu method demonstrates that the group of D50N+DOX has the highest inhibition of cell proliferation,followed by the group of A88V+DOX,and the group of V37E+DOX has the lowest inhibition of cell proliferation.The result of Caspase3/7 enzyme activity method shows that apoptosis of HaCaT cells increases in the group of D50N+DOX,however decreases in the group of A88V+DOX and the group of V37E+DOX compared with the group of NC+DOX(blank group)and the group of WT+DOX(control group).The result of Senescence-associated beta-galactosidase dyeing method illustrates that the number of senescent cells in each group is relatively small(some fields of vision do not show senescent cells),scilicet there is no obvious senescent cells in each group.Conclusions Although the cases of D50N mutant type are extremelly rare,this kind of mutant type has greater value to research than A88V and V37E mutant type.Owing to the particularity of D50N mutant type in the cellular functional experiment,it illustrates that it is likely to be related to the complex symptoms or even some oppsite symptoms of hidrotic ectodermal dysplasia,such as the decks of most patients become thicker,however the nails of few patients become thinner and more crisp.Further animal experiments are needed in order to find more symptoms.