Research On Interfering The Immune Recognition Mediated By Nanometer Vector

In this study,cation polymeric liposome vector of nanometer and small hairpin RNA(shRNA)plasmid vectors targeting rat class Ⅱ major histocompability complex transactivator(CⅡTA)and myeloid differentiation factor 88(MyD88)genes were constructed.Both nanometer liposome and shRNA plasmid were coupled to form the nanometer vectors containing targeted shRNA for further research.In vitro transfection of rat myeloid dendritic cell(DC)was performed to investigate the inhibiting effect on CⅡTA,MyD88 and class Ⅱ major histocompability complex(MHC-Ⅱ)expression by nanmometer vector mediated shRNA interfering.Mixed lymphocyte culture and lipopolysaccharide(LPS)stimulate experiment were done to evaluate the inhibiting effect on immune recognition by the object genes silencing and the underlying mechanism was investigated.Part I Construction of Nanometer Vector Containing shRNA and in vitro Transfection ResearchObjective:To construct nanometer vectors containing targeted shRNA and determine the in vitro transfection plan to HCT-116 cell line by transfection efficiency.Methods:Cation polymeric liposome vector of nanometer and small hairpin RNA(shRNA)plasmid vectors targeting rat class Ⅱ major histocompability complex transactivator(CⅡTA)and myeloid differentiation factor 88(MyD88)genes were constructed,coupled and characterized.MTT test was done to identify the biological safety of nanometer vector.HCT-116 cell line was cultured for transfection.There were totally 8 groups designed for transfection:control group,plasmid group,Lipofectamine group and nanometer groups with different weight ratio(1:0.5,1:1,1:2,1:3 and 1:4).EGFP expression was detected by flow cytometry(FCM)and fluorescence microscope to determine the transfection efficiency.Results:Cation polymeric liposome vector of nanometer and small hairpin RNA(shRNA)plasmid vectors were successfully constructed and coupled.Nanometer vector was identified as a safe(IC50=7.605×10-2g/L),low cytotoxic and stable vector.The optimal weight ration for coupling is 1:2.Among transfection groups,nanometer group with the optimal weight ratio showed highest transfection efficiency(28.19±5.38%)and was better than commercial Lipofectamine group(P<0.01).Conclusion:Cation polymeric liposome vector of nanometer was proper as a promising gene transfer vector for simple preparation,safety and low immunogenicity.Nanometer vectors showed obvious superiority on HCT-116 cell line transfection in vitro and could be used for further study of the inhibiting effect of CⅡTA and MyD88 gene expression by RNAi.Part Ⅱ Research on the Inhibiting Effect of the Objective Gene Expressions Mediated by Nanometer VectorObjective:To investigate the inhibiting effects of nanometer vector containing shRNA against CⅡTA and MyD88 genes and the regulation of MHC-Ⅱ expression by CⅡTA.Methods:In vitro culture of the rat myeloid DC was performed.There were totally 6 groups for DC transfection of CⅡTA and MyD88 respectively:control group,polymeric liposome vector of nanometer control group,HK nanometer vector control group and three CⅡTA or MyD88 nanometer vector experimental groups.Real time QRT-PCR,FCM and Western blot were performed to analyze the expression changes of CⅡ TA,MHC-Ⅱ and MyD88.Results:Sufficient rat myeloid DC were obtained by in vitro culture.Compared to 3 groups,the transcription levels of CⅡ TA and MHC-Ⅱ and the expression level of MHC-Ⅱ were significantly inhibited in all three CⅡ TA nanometer vector experimental groups(P<0.01).There was positive correlation between the expression of CⅡ TA and MHC-Ⅱ.The transcription and expression levels of MyD88 were significantly inhibited in all three MyD88 nanometer vector experimental groups(P<0.01).Among the experimental groups,CⅡTA-2 nanometer vector and MyD88-1 nanometer vector showed the strongest inhibiting effect on targeting gene expression.The transcription levels of CⅡTA and MHC-Ⅱ were 0.12±0.04 and 0.10±0.04 and the expression level of MHC-Ⅱ was 22.08±3.07%.The transcription and expression levels of MyD88 were 0.11±0.04 and 0.11±0.05.Conclusion:The expressions of CⅡTA and MHC-Ⅱ in rat DC were inhibited after cⅡTA nanometer vector transfection.The regulating effect on MHC-II expression by CIITA was confirmed by the significant positive correlation between them.The expressions of MyD88 was inhibited after MyD88 nanometer vector transfection.CⅡTA-2 and MyD88-1 nanometer vectors showed strongest inhibiting effects and could be applied for further study on interfering immune recognition.Part Ⅲ Research on Interfering the Immune Recognition Mediated by Nanometer VectorObjective:To evaluate the interfering effects on immune recognition by CⅡTA-2 and MyD88-1 nanometer vectors and explore the underlying mechanism.Methods:Rat DC transfected with CⅡ TA-2 nanometer vector was used to perform mixed lymphocyte culture.There were totally 5 groups:positive control group,negative control group,polymeric liposome vector of nanometer control group,HK nanometer vector control group and CⅡ TA-2 nanometer vector experimental group.MTT test was done to detect cell stimulation index.Enzyme-linked immunosorbnent assay(ELISA)was done to detect INF-y and IL-2 expressions in supernate fluid.FCM was done to detect T cell subset examination.Rat DC transfected with MyD88-1 nanometer vector was used to perform LPS stimulate experiment and the groups design as above.Expression of MyD88 was detected.Expressions of INF-γ,IL-2 and MHC-II were also detected.DC was then used for continuous mixed lymphocyte culture and the cell stimulation index was detected.Results:In mixed lymphocyte culture,the cell stimulation index of CⅡTA-2 nanometer vector experimental group decreased significantly compared to positive control group,polymeric liposome vector of nanometer control group and HK nanometer vector control group(1.15±0.14,P<0.01).Similarly,INF-γ and IL-2 expressions decreased(INF-γ42.39±7.68 pg/mL,IL-2 67.83±18.35 pg/mL;P<0.01)and CD4 positive T cells and CD4/CD8 ratio both decreased(48.38±8.63%,P<0.01;1.89±0.26,P<0.01).In LPS stimulate experiment,mRNA and protein expression of MyD88 of MyD88-1 nanometer vector experimental group both decreased significantly compared to positive control group,polymeric liposome vector of nanometer control group and HK nanometer vector control group(0.22±0.08,0.16±0.04;P<0.01).Similarly,INF-γ and IL-2 expressions decreased(INF-γ32.49±5.65 pg/mL,IL-2 21.08±4.13 pg/mL;P<0.01)and MHC-Ⅱ protein expression decreased(45.21 ±5.09%,P<0.01).The cell stimulation index of MyD88-1 nanometer vector experimental group also decreased obviously compared to positive group(1.23±0.11,P<0.01).Conclusion:Silence of CⅡTA and MyD88 gene expression mediated by nanometer vectors containing shRNA inhibited immune recognition efficiently.The mechanism maybe related to reduced DC immunogenicity and interrupt of signal transduction pathways.Depression of subsequent T cell stimulation and activation and stop of efficient antigen presentation progress could lead to inhibition of immune response and be helpful to control rejection reaction.