Construction And Identification Of ImDC Interference Vector Of CD80/CD86 Gene RNA Interference Vector In Rats

Currently, liver transplantation is the primary method in the treatment of advanced liver disease. But the acute or chronic rejection response after the transplantation is the key factor that influences the recovery and survival of the recipient. Immunosuppressant can effectively inhibit the rejection response, but long-term uptake of the inhibitor will cause adverse reactions eventually. Induction of transplant tolerance can overcome the immunological rejection, improve recipient’s quality of life, and it may be the best way to overcome the rejection response after organ transplantation. CD80 and CD86 are the most important co-stimulatory molecules presented on dendritic cells’surface. Combined with the cell’s surface molecule, they synergistically induce the naive lymphocytes to the memory cells or the effector cells, thereby inducing cell proliferation and differentiation, which plays important roles in the process of co-stimulation. By suppressing the expression of CD80 and CD86 in the DCs’ surface, and blocking the co-stimulatory pathway, donor-specific immune tolerance may be induced. Studies in recent years indicated that some of the double-stranded RNA which includes specific DNA can degrade mRNA, thus efficiently inhibits the expression of specific gene in vivo, and induces phenotype deletion of specific gene, which is known as RNA interference. As RNAi technology is featured with high-efficiency and specificity, it has become an indispensable tool in the functional genomics research in post-genomic era. CD80 and CD86 gene loaded imDC specific lentiviral vectors can be built with RNA interference, and their impact on imDC’s expression of surface molecule can be observed ex vivo. In theory, once the mRNA sequence of a particular gene is detected, RNAi can be applied to the suppression of this gene, which can even achieve the result similar to gene knockout. This provides a new approach in inducing immune hyporesponsiveness or even immune tolerance by inhibiting co-stimulation. RNAi can knock out the CD80/CD86 genetic pathway in co-stimulation in donor’s DCs. If costimulatory molecules mRNA’s expression in this process can be inhibited, thus suppress the co-stimulation signal, to induce the anergy of reactive T cells, the purpose of the anti-rejection can be achieved. However, CD80 and CD86’s interference effect is directly influenced by the different interference efficiency in different sites of action. Therefore, we have designed three interference fragments with different sites of action, and find out the best one through comparison, to prepare for further study on inhabiting co-stimulation by donor’s DC that is pre-processed with recombinant lentivirus ex vivo and on immune tolerance after liver transplantation in rat model.Objective (s):To build CD80 and CD86 loaded imDC specific lentiviral vector, observe the impact on immature bone marrow derived dendritic cells’expression of surface molecule in rat model ex vivo.Methods:to screen and select potential target sequence of the RNA interference of CD80 and CD86 in imDC in rat model, compound the lentiviral vector. To infect the rat’s bone marrow derived (imDC) cultured ex vivo with lentivirus, inspect the infection efficiency with fluorescence microscope and flow cytometer, and detect changes of the protein volume of the CD80 and CD86 genes with Western Blot, according to the difference of the CD80 and CD86’s expression inspected with flow cytometer pre-and post-infection.Results::recombinant plasmid shRNA PCR, the sequencing of the shRNA’s Oligo DNA indicates that the structures of lentiviral vector LV-GFP-shCD80 and LV-FGP-shCD86 are correct; After lentivirus’s infection to imDC, flow cytometry indicated declines in expression of CD80 and CD86 in three groups, comparing with imDC in the other two control groups, statistically significant difference is indicated (p<0.01) while expressions of OX62 and MHC-Ⅱ are not affected. Western Blot prompts that after lentivirus’s transfection, significant declines in protein’s expression volume of imDC’s CD80 and CD86 gene are noticed comparing to that in the negative control group, in which the volume of LV-GFP-shCD80(c) and LV-GFP-shCD86(c) declines the most.Conclusion:the CD80 and CD86 gene RNA interfered lentiviral vector in rat model is built correctly, CD80 and CD86’s expression on imDC’s surface can obviously be inhibited by the interference sequencing in three groups, among which, the shCD80(c) and shCD86(c) provide the best outcomes.