Pathogenesis Research Of A Novel Rare Single Nucleotide Mutation Of IL36RN Gene Founded In Children With Generalized Pustular Psoriasis

Part ? Centrifugation Increases the Gene Transfection Efficiency of Ha CaT CellsObjective: HaCaT cells are spontaneous human immortalized epidermal cell lines that are commonly used in the study of skin function and disease but the efficiency of conventional liposome transfection of the cells is extremely low.This study is aimed to investigate whether centrifugation can increase the efficiency of liposomal transfection of HaCaT cells.Methods: Lipofectamine? 2000 was used as a liposomal transfection reagent,and pEGFP-C1 was used as a plasmid,and HaCaT cells were transfected by conventional method and centrifuged method respectively.Transfection efficiency was observed by inverted fluorescence microscope,quantitatively determined by flow cytometry.Cell proliferation activity was measured to determine whether centrifugation affect cell viability.The luciferase reporter assay and Western Blot were carried out to detect the effects of centrifugation on the expression of the target genes.Results: The transfection efficiency of centrifugation treatment was higher than that of the conventional treatment.The difference was statistically significant.Luciferase reporter gene assay and Western Blot experiment further confirmed that the centrifugation after transfection can promote the expression of the target gene,which has high practicability.Conclusion: The transfection efficiency of HaCaT cells can be increased by centrifugation after conventional transfection procedures.Part ? Identification of IL36 RN Gene Mutation and Its Pathogenic Mechanism ResearchObjective: Generalized pustular psoriasis(GPP)is a rare but severe inflammatory skin disease that is sometimes life-threatening.The main purpose of this article is to investigate the pathogenic mechanism of newly discovered single nucleotide mutations in the IL36 RN gene.Methods: Genomic DNA was extracted from the peripheral blood of the patient and her parents.DNA fragments containing all the coding regions of the IL36 RN gene and adjacent flanking intron sequences amplified by PCR were sequenced to find nucleic acid mutations.The effects of the IL36 RN gene mutation on the protein and m RNA were checked by Western Blot and q PCR experiments respectively,Wild-type and mutant IL36 RN gene expression vectors were constructed and transfected into cultured cells.Finally we used protein structure modeling and molecular dynamics simulation to further elucidate the possible mechanisms of the mutation.Results: In this article,we identified one previously unreported single nucleotide missense mutation c.59A>G of IL36 RN gene.Western Blot experiments showed that the mutation reduced the protein expression of IL36 RN gene,while q PCR experiments found that the mutation of c.59A>G did not reduce the transcription of IL36 RN gene;The protein structure analysis by modeling and molecular dynamics simulation suggested that mutation of IL-36 RN caused the IL36 Ra protein in an unstable state.Conclusion: we concluded that missense mutation p.Tyr20 Cys can cause the IL-36 Ra degradation and make variant c.59A>G to be a loss-of-function and pathogenic mutation,which underlies the genetic basis of the patient's GPP.