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Adenovirus-Mediated Gene Transfer of Antisense CⅡTA Ameliorate Experimental Autoimmune Myocarditis

Author: CaiGang
Tutor: ShenZuo；LiWenJie
School: Second Military Medical University
Course: Clinical Laboratory Diagnosis
Keywords: MHC-Ⅱ C Ⅱ TA Antisense gene Adenovirus Experimental autoimmune myocarditis
CLC: R542.21
Type: Master's thesis
Year: 2004
Downloads: 47
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Abstract

Major histocompatibility complex (MHC) Ⅱ molecules exogenous antigens can be presented to T helper cells, caused by the activation of antigen-specific T cells. Constitutive expression of MHC-Ⅱ molecules is limited to \Both constitutive and inducible expression of MHC-II molecules, MHC-II molecules trans-activating factor (C Ⅱ TA) are absolutely necessary. In recent years, the research suggests that C Ⅱ TA is to control the master regulator of MHC-Ⅱ gene expression to determine the existence and degree of expression of MHC-Ⅱ molecules, and also determines the nature of the immune response. The ideal target as the impact of a large gene family of a single gene or protein, C Ⅱ TA MHC-II molecules presenting antigen process intervention in autoimmune injury and transplant rejection process immunosuppressive intervention. The antisense nucleic acid technology is used according to the principle of complementary base closed target genetic material (mRNA or DNA) specific complementary nucleic acid fragment of gene expression. Antisense RNA (antisenseRNA) is an antisense technique, its down the exact mechanism of gene expression is unknown, may be combined with mRNA, the formation of dimers and interfere with the synthesis of specific proteins, this binding reaction can occur in the transcription completed to the various stages of translation initiation, including RNA splicing, transport and translation initiation complex binding to the initiation codon. Antisense nucleic acid fragments of recombinant adenovirus gene transfer vector to overcome its poor stability, poor cell penetration shortcomings, the feasibility and effectiveness of antisense nucleic acids for gene therapy. Experimental autoimmune myocarditis (experimental autoimmune myocarditis, EAM) due to human myocarditis similar pathological features of the disease process, so often as to study the of myocarditis pathogenesis of animal model. EAM is a T cell-mediated myocardial cell degeneration, necrosis, fibrosis, and accompanied by a large number of mononuclear cell infiltration of the pathological features of autoimmune inflammation. MHC-II molecules in myocardial tissue abnormal expression of a large number of local infiltration of CD4 T cells and macrophages, pre-inflammatory cytokine secretion increased, leading to anti-cardiac myosin activation of autoreactive T cells is mainly caused by normal myocardial injury. reasons. Therefore, it is possible by targeting inhibit the expression of C Ⅱ TA, lowered the expression of MHC-Ⅱ molecules and the autoantigen peptide delivery was to achieve the purpose of the prevention of EAM. Designed antisense fragment of mouse p of IV type C Ⅱ TA mRNA as a template, a fragment of a total length of 431-bp, covering the initiation codon 22 bp upstream reading frame 5 'end of the 409 bases, and also contains the first between the exon and the second exon splice sites; antisense fragment of adenovirus expression vector (pAdEasy-1 system) is constructed using conventional molecular biology methods; adenovirus vector generated HEK293 cells packaging containing trans 3 lt high titer adenovirus; WP = 7 gt; Thesis Chinese summary justice fragment recombinant adenovirus Ad-C Ⅱ TA, purified by cesium chloride density gradient centrifugation; using a variety of methods to quantitative detection of recombinant adenovirus the active evaluation; and Ad-C Ⅱ TA HeLa cells were infected with P388D1 cells, the observed inhibition of the expression of inducible and constitutive MHC-II molecules; induction experiments with purified porcine cardiac myosin immunization of Balb / c mice, autoimmune myocarditis generation of animal models, and 0 to 2 days and 14 to 16 days after immunization by intravenous injection of recombinant adenovirus observe the prevention and treatment effect of Ad-C Ⅱ TA experimental autoimmune myocarditis. The first part of the construction of recombinant adenovirus vector carrying mouse MHC-Ⅱ molecule trans-acting factor gene antisense fragment used in this part of the conventional molecular biology methods, start by IFN-γ-stimulated murine peritoneal mononuclear macrophages extracted total RNA, anti-transcribed to obtain cDNA the mouse p Ⅳ C Ⅱ TA Primer3 software design primer PCR amplified target fragment obtained by the pMD18-T vector cloning sites, loaded into the recombinant shuttle plasmid pAdTrack-CMV. Pme Ⅰ fragment was digested with the purpose of the shuttle plasmid pAdTrack-CMV recombinant adenovirus skeleton plasmid pAdEasy-1, transformed into E. coli DH-5α, homologous recombination occurs recombinant adenovirus carrying antisense fragment skeleton the purified backbone plasmid identified, cut enzyme Pac Ⅰ linearization skeleton, HEK293 cells transfected with recombinant adenovirus particles Ad-C II TA. The partially constructed recombinant adenoviral vector for antisense inhibition of C Ⅱ TA gene expression has laid a foundation for the prevention and control of the expression of MHC-Ⅱ molecules autoimmune disease animal models, quantitative lowered. The part of the second part of the recombinant adenovirus purification and quantitative detection by flow cytometry, phage plaque formation test, half of the tissue lesions, and real-time quantitative PCR, HPLC OD260 colorimetry CsCl double density gradient ultracentrifugation purified recombinant adenovirus infection activity with the actual number of virus particles. Does not exceed 102 times the average gap between the two sets of methods, and results between the two groups within the method has obvious comparable, indicating that the purified recombinant adenovirus with more stable properties. The method for determination of infection activity is often a lack of objective criteria, and time-consuming and labor-intensive, prone to causing the error, and the method of determination of the number of actual virus particles does not reflect the activity of virus infection, we recommend to use in viral titer was determined joint evaluation of a variety of methods. Since we purified viral activity is relatively high, in order to maintain the reproducibility of subsequent experiments, we used the actual number of viral particles as a dose standards. Adenovirus third part mediated antisense C Ⅱ TA gene transfer inhibition of MHC-Ⅱ class molecule expression of in vitro studies This section first of all use of recombinant human or mouse's of IFN-γ, respectively, the cell strain HeLa and P388D1 carried stimulation, observation of IFN-γ-induced MHC role - II molecule expression. Found that recombinant human IFN-γ can induce HeLa cells expressing MHC-II molecules, with dose increases its expression is elevated. But no apparent induction of recombinant murine IFN-γ expression on P388D1 cells MHC-Ⅱ molecules. Were used recombinant adenovirus

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CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Heart disease > Myocardial diseases > Myocarditis
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