Construction The Lentiviral Vector Targeting Human DC-SIGN Gene And The Functional Identification

DC-SIGN(dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintergrin, DC-SIGN) is a new kind of lectin receptors on the surface of dendritic cells (DCs). The main functions of DC-SIGN are regulating the adhesion and migration of DCs, activating the naive T lymphocytes, priming the immune response , the immune escape of pathogens and so on. It is the main receptor of Mycobacterium tuberculosis(Mtb) on the surface of DCs . Mtb can combine tightly with DC-SIGN by its cell wall component-ManLAM, thus cause the maturation inhibition of DCs. DCs plays a central role in the anti-TB immune response as it is the only antigen-presenting cell to activate the naive T cells and priming the incipient immune response. However, only the mature DCs can activate the naive T lymphocytes. RNAi can down regulate the expression of targeting gene efficiently and specificly. Therefore it has broad applications in the field of gene function studies and gene therapies. The Lentiviral vector is one of the vehicles which is uslally used in the RNAi technology . It can bring their own gene pieces to integrate the host cell genomes, and then express small interfering RNA stably, thus suppress the expression of targeting gene for a long-term.At first we choosed four targeting sequences according to the mRNA sequences of human DC-SIGN gene , and then designed and synthesized double-stranded DNA. We selected the effective RNAi targeting sequences after PCR amplification, DNA sequencing as well as exogenous screening. The human DC-SIGN gene lentiviral vector was constructed and packaged. We transfected the endritic cells which differentiated from human peripheral blood mononuclear cells(PBMCs)with the RNAi Lentiviral expression vector. The expression level of DC-SIGN was detected by flow cytometry,RT-PCRand Western Blot. The mature markers of DCs were detected by flow cytometry. The levels of IL-12 and IL-10 in the supernatant were measured by enzyme-linked immunosorbent assay(ELISA). The ability of DCs to stimulate the proliferation of CD4+T lymphocytes was measured by mixed lymphocyte reaction(MLR). Through the above studies, we could clarify that whether RNAi mediated by the lentiviral vector can effectively inhibit the expression of DC-SIGN and influce the maturation and function of DCs.PCR and DNA sequencing demonstrated that the inserted sequences were correct . Two effective targeting sequences were selected by exogenous selection. The titer of concentrated virus was 1×10 9 T U/mL. The expression level of DC-SIGN detected by flow cytometry ,RT-PCR and Western Blot in the interference group was significantly lower than the control groups. The maturity degree of DCs had no significant difference, although the expression levels of DC-SIGN were different. There were no significant differences in the concentrations of IL-12 and IL-10 in the DCs culture supernatant of all groups. DCs in each group had the similar abilities to stimulate CD4+T lymphocytes cell proliferation.Our study have built a new way to transfect the DCs efficiently and stably which can inhibit the expression of DC-SIGN on the surface of DCs in vitro. At the same time, the DCs which are transfected by our lentiviral vector can maintain its own pattern, growth modality, maturity Phenotype and immunological function. It is a useful tool to regulate the expression level of DC-SIGN molecule. The results provide us a foundation to furtherly study the effect on the downstream anti-TB immune response through regulating the expression level of DC-SIGN.