In Vitro Study Of The Effect Of Silencce Of CD40 Gene Expression In Denytritic Cells By RNAi On Organ Transplant Rejection

As a kind of powerful antigen presenting cell(APC),dendritic cells(DCs) are investigated more and more with aidding T and B lymphocyte's immunological function and found that they have important means in the happening and develpping process of transplantation reject reaction, rheumatoid disease,tumor, allergy and autoimmune disease.In the local tissue of these diseases the DCs have change with quantity and or or surface marks,then that leads to produce of autoantibody,form of rheumatoid factor or defect of local immune function.In present,the more using of DCs is immune of anti-tumor and anti- transplantation reject reaction,but the research with utilizing RNA interference(RNAi) silences DCs to inhibit actue immunol- ogical reject reaction of transplantation is fewer.RNA interference(RNAi) is technolagy of gene silence of posttrans- cription,small or short inference RNA(siRNA) can target some kinds of monitoring progress of posttranscription, identify mRNA which has the homology sequence,then specifily cut the mRNA and obstruct its interpretation function.The investigation uses the RNAi silence CD40 of DCs which gets from the bone marrow of the rats,which effects the expression of the DCs.Through the test in vitro to investigate the inhibitional contribution of lymphocyte of spleen through interferencing the expression of CD40. Object To investigate RNAi silenceing CD40 gene treats actue immunological reject reaction of transplantation,and commit some contributions for treatment of the actue immunological reject reaction of transplantation.Method1. the cultivation of DCsDCs were cultivated as regular method. Photos of DCs'growth were taken every day. The activation and maturation markers of dendritic cell were detected by flow cytometer at 12th day.2. preparation of recombinant virus vector expressing CD40 siRNAUsing CD40 gene sequences from GenBank, selected suitable target site, synthesized loignucleotides as DNA template encoding CD40 siRNA, annealed and ligated into lentivirus expressing vector to construct recombinant virus vector expressing CD40 siRNA.3. Construction of T293 cell which expressed CD40 gene and RNAi this cell with recombinant lentivirus vector expressing CD40 siRNAT293 transfection cell expressing CD40 gene were constructed as a toll cell to explor the method of RNAi. The T293 cell which expressed CD40 gene were transfected with recombinant lentivirus vector. To determine the expression of the CD40 after transfection, Western blot analyses with samples extracted from transfected and control cells were performed.4. Expression of CD40 gene in DCs which are interferenced by RNAi is determined by Western blot.The CD cells were transfected with recombinant lentivirus vector. To determine the expression of the CD40 after transfection, Western blot analyses with samples extracted from transfected and control cells were performed.5. one-way mix lymphocyte reactionsplenocytes cells of the rat were isolated, and were mix-cultured with DC interferenced by RNAi. Proliferation of splenocytes were assayed by 3H-TdR incorporate method. ELISA for IFN-γ,IL-2,IL-4,IL-10 were performed to determine the secretion of related cytokine. Statistical analysisData were wxpressed as mean±SE. Comparison between groups was performed with student's t testχ2 analysis using a sigmastat statistical software package(SPSS,Chicatgo,IL). P<0.05 was taken as showing siginificance.Results①DCs are successfully induced and cultivated from the bone marrow cells of the rat in vitro. The condition of DCs'activation and maturation was well and these DCs could be used in the following experiment.②The recombinant lentivirus vector expressing CD40 siRNA was constructed, and confirmed by restriction enzyme digest and sequence analysis.③T293 transfection cell expressing CD40 gene were constructed. Transfection with recombinant lentivirus vector inhibited the CD40's expression of T293 transfection cell. The results from Western blot for the samples from T293 cells after transfection demonstrated that the recombinant lentivirus vector expressing CD40 siRNA could specifically reduce CD40 expression. ④Transfection with recombinant lentivirus vector inhibited the CD40's expression of DCs. The results from Western blot for the samples from DCs after transfection demonstrated that the recombinant lentivirus vector expressing CD40 siRNA could specifically reduce CD40 expression.⑤The results of 3H-TdR incorporate method showed that Proliferation of splenocytes in CD40 siRNA groups was significantly lower than that of control groups. ELISA exhibited that the cytokine IFN-γ,IL-2,IL-4,IL-10 in siRNA groups were significantly lower than those in control groups.Conclusion :1. The method of inducing DCs from bone marrow of rat in vitro is safe and convenient.2. The technology of RNAi can interferences the expression of CD40 in the second signal pathway of DCs,and absence of the stimulus signal will lead to inability or apoptosis of T lymphocyte.3. Through the technology of interference CD40 of DCs,two RNAi sequences with interference well are successfully got, and the result consistents with what is expected. The test in vitro demonstrate that the ability of activating immune cell of DCs which are interferenced by RNAi obviously decreases.