Adenovirus-Mediated Gene Transfer Of Antisense Câ…¡TA Ameliorate Experimental Autoimmune Myocarditis

Major histocompatibility complex (MHC) class Ⅱ molecules are thepredominant presenters of exogenous antigens to T helper cells. Constitutiveexpression of MHC-Ⅱis restricted to "professional" antigen-presenting cells but canbe induced on various tissues by gamma interferon(IFN-γ). The class Ⅱ transactivator(CⅡTA) expression appears to be an absolute requisite for expression of MHC-Ⅱ,whether constitutive or inducible. Studies in recent years suggest a view of CⅡTA asa master regulator controlling expression of class Ⅱ MHC genes. The regulation ofCⅡTA largely determines the presence or absence of MHCⅡand its degree ofexpression and thus the nature of the immune response. As CⅡTA is a single gene orprotein that affects a large family of genes, it represents an ideal target forintervention in the class Ⅱ antigen presentation pathway, for immune suppression intransplantation and autoimmunity. Antisense nucleic acid techniques are a group of methods using single-strandedcomplementary sequences with specificity assigned by Watson-Crick base-pairingtargeted specific messenger RNA or DNA to block expression of target gene. Theexact mechanism of antisense RNA technique which is one of the antisense nucleicacid techniques remains unclear. Generally, these molecules block gene expression byhybridizing to the target mRNA, resulting in subsequent double-helix formation. Thisprocess can occur at any point between the conclusion of transcription and initiationof translation, or even possibly during the translation. Disruption of splicing, transport,or translation of the transcripts are all possible mechanisms, as is stability of transcript.Antisense nucleic acid drawing assistance from adenovirus vector will overcome thedefects of stability and penetration, and promote the technique's effectiveness andfeasibility in the field of gene therapy. Experimental autoimmune myocarditis (EAM) has proved to be a highly usefulanimal model of human inflammatory heart disease because its pathologicalcharacters and progression are similar to clinical autoimmune myocarditis. EAM is akind of organ destructive autoimmune disease mediated by T cell immune, and 7硕士论文 英文摘要charactered by cardiac myocyte degeneration, necrosis, fibrosis, as well asinfiltration of monocnuclei cells. Activation of autoimmune responsed T cells whichinduced by abnormal expression of MHCⅡ, infiltration of CD4 T cells and +macrophages, and increasing of pre-inflammatory factors is the major cause of heartlesion. Thus, it is imaginable that down-regulation of MHCⅡexpression andpresenting auto-antigen peptide by target suppression of CⅡTA expression canprevent the both induction and the progressive of EAM. In this study, we designed a fragment of mouse type pⅣ CⅡTA cDNA inantisense orientation using mRNA of CⅡTA as template. The primers were designedbased on 83~513 nucleotides segment in 5' end of CⅡTA cDNA so that the interestedfragment contained 431 base pairs which included AUG codon in 101st nucleotide aswell as the splicing site between the first and the second exons. Through routinemolecular biological techniques we constructed recombinant adenoviral vectorcontaining the antisense fragment. Recombinant adenoviruses were generated bypackaging of HEK293 and purified by velocity density gradient centrifugation incaesium chloride solutions. Then we conducted in vitro gene transfer, infecting HeLaand P388D1 with recombinant adenoviruses respectively, in order to observeinhibition of constitutive or inducible CⅡTA gene expression by introducedcomplementary antisense RNA of CⅡTA and consequent downregulation of class ⅡMHC molecules expression. After production of EAM model by immunized withmyosin, recombinant adenovirus was injected to mice's veins at day 0~2 or day 14~16to evaluate the value of the virus...