Study On Inhibition Of Class Ⅱ MHC Molecules Expression By Gene Transfer Of Antisense CⅡTA

Major histocompatibility complex (MHC) class II molecules are the predominant presenters of exogenous antigens to T helper cells. Constitutive expression of class II MHC is restricted to "professional" antigen-presenting cells but can be induced on various tissues by gamma interferon. The class II transactivator (CIITA) expression appears to be an absolute requisite for expression of class II MHC,whether constitutive or inducible. Studies in recent years suggest a view of C II TA as a master regulator controlling expression of class II MHC genes. The regulation of C II TA largely determines the presence or absence of class II MHC and its degree of expression and thus the nature of the immune response. As C II TA is a single gene or protein that affects a large family of genes, it represents an ideal target for intervention in the class II antigen presentation pathway, for immune suppression in transplantation and autoimmumty.Antisense nucleic acid technique is a method using single-stranded complementary sequences with specificity assigned by Watson-Crick base-pairing targeted specific messenger RNA or DNA to block expression of target gene. The mechanism is that the introduced complementary oligonucleotides can bind to the corresponding mRNA or double-stranded DNA in genome and form partial double-stranded molecules or triple-stranded nucleic acid molecules by sequence-specific and nonsequence-specific antisense action, thus the target gene will be orientationally blocked and expression of the target inhibited so that therapeutic effect could be attained.In this study, we designed a fragment of human C II TA cDNA in antisense orientation using mRNA of C II TA as template. The primers were designed based on 94-500 nucleotides segment in 5' end of CIITA gene so that the interested gene contained 407 base pairs which included two AUG codons in 1 16 and 188 nucleotides as well as the splicing site between the first and the second exons. Through routine molecular biological techniques we constructed recombinant adenoviral vector containing the antisense fragment. Recombinant adenoviruses were generated by packaging of HEK293 and purified by velocity density gradient centrifugation in caesium chloride solutions. Then we conducted in vitro gene transfer, transfecting P388D1 with recombinant eukaryotic vector and infecting HeLa with recombinantadenoviruses respectively, in order to observe inhibition of constitutive or inducible CII TA gene expression by introduced complementary antisense RNA of C IITA and consequent do wnregulation of class II MHC molecules expression.Results demonstrated that after P388D1 cells with constitutive expression of class II MHC molecules were transfected by eukaryotic vector containing CII TA antisense fragment and selected by antibiotics, the positive rate of I-Ad molecules expressed on P388D1 was decreased by 65.7% compared with the control meanwhile mean fluorescent intensity by 40.1%, also after HeLa cells with inducible expression of class II MHC molecules were infected by recombinant adenoviruses containing CII TA antisense fragment, the positive rate of HLA-DR molecules expressed on HeLa induced by IFN- Y was decreased by 5~24%(mean 15%) compared with the control meanwhile mean fluorescent intensity by 58-83 % (mean 74%). The results of inhibition of T cells activation assay indicated that activation of T lymphocytes which mainly recognize the different compatibility of class II MHC molecules was inhibited in different degree due to the inhibited inducible expression of class II MHC molecules on HeLa by recombinant adenoviruses containing CII TA antisense fragment, showing as decreases in the positive rate and mean fluorescent intensity of CD25 and CD69 expression, which are both signs of activated T cells. In one of the cases, the positive rate of CD25 was decreased by 17.1% meanwhile mean fluorescent intensity by 50.8%, also the positive rate of CD69 was decreased by 16.7% meanwhile mean fluorescent intensity by 56.7%. In conclusion, antisense C II TA can partially...