Study In Anti-tumor Immunity Of Sensitized Dendritic Cells Induced By Tumor Cells With Transfection Of CD40L

Considering the limitations of chemotherapy and radiotherapy in the treatment of tumors, it seems more reasonable to take immune therapy which will activate the potential anti-tumor effects of their own bodies in tumor patients. Immunity therapy dose not only cooperate well with chemotherapy and radiotherapy to fight against tumor, but also gives the patients an effective adjustment and support for the functional states of their bodies. Therefore, people have great expectation of immune therapy. With the development of research on immunology, especially the deeper understanding of the role of dendritic cells and some cytokines in immune response, tumor immune therapy has got new developments in recent years.Vaccines are critical for tumor immune therapy, among which DC vaccine wins great attention. And present researches are focusing on how to make the tumor antigen presented by DC more specific with better potency in activating T effector cells. CD40 and ligand CD40L(CD154)exist on the membrane of different cells in immune system, and they play a pivotal role in modulating the body fluid and cell immune responses by interacting with each other. Recent researches indicate that the CD40 signals can be activated by treatment of malignant tumors. The combination of CD40L with CD40 on the membrane of tumor cells can directly inhibit the proliferation of tumor cells and increase their sensibility to anti-tumor praeparatums in certain condition. Moreover, it can enhance the anti-tumor immunity of the patients. Our study aims to construct a DC vaccine costimulated by CD40L and homogenous tumor components on the basis of the special function of DC and CD40L in immune response.Methods1.Construction of h-CD40L expression system(1)First isolated T lymphocytes from human blood with cell isolation and culture technique, then extracted the total RNA of T cells after their activation.(2)RT-PCR was performed for the reverse transcription and Amplification of CD40L gene fragment.(3)A CD40L recombination cloning vector was constructed with purified CD40L gene fragment and pMD-18-T vector, then it was transformed into DH5αcompetent bacteria followed by Ampicillin and blue/white screening of the transformed bacteria as well as bacteria PCR and identification of cloning vector with DNA sequencing.(4)First extracted recombinant cloning vector pMD-18-T-CD40L and eukaryotic expression vector pcDNA3.1(+) plasmid from corresponding transformed bacteria with plasmid extraction kit, then they were digested with enzyme, purification and connection of the digested products were performed to construct an eukaryotic expression vector pcDNA3.1-CD40L which was then transformed into TOP10 competent bacteria, followed by screening of the transformed bacteria with Ampicillin, as well as bacteria PCR , double enzyme digestion by NheⅠa nd KnpⅠand identification of the eukaryotic expression vector pcDNA3.1-CD40L with DNA sequencing .2 Transfection of CD40L expression vector into tumor cells.(1) After its thawing and culture, H446 cells were inoculated into 6 well plates followed by transfection of pcDNA3.1-CD40L with liposome.(2)Transcription level of CD40LmRNA in H446 transfected cells and the membrane binding rate of PE-labelled CD40L antibody were detected with RT-PCR and FCM respectively.(3)Linear and circular pcDNA3.1-CD40L recombinant plasmids were transfected into the following three cell lines: SK-hepⅠ, H446 and H460,followed by its expression rate analysis.(4) Primary cells from surgical pulmonary carcinoma tissue were cultured with primary cell culture technique, followed by transfection of pcDNA3.1-CD40L with liposome and CD40L expression detection by FCM.3.Test of in vitro immunity function of DC sensitized by tumor cells expressing CD40L.(1)DC induction and homogenous T cells culture were performed according to cell culture technique.(2)DC induced for 7 days were sensitized with special sensitizing agents made of H460 after its freeze thawing or 1% formalin treatment of G418 screening after CD40L transfection.(3)Expression of cell surface molecules(CD83,CD86,MHC-) on sensitized DC were detected with FCM, whose results were compared with that of un-transfection group.(4)Secretory volume of IL-12 in sensitized DC un- transfection group, empty vector group and transfection group were detected and compared with each other by ELISA.(5)Sensitized DC were co-cultured with homogenous T cells for 5 days after its inhibition with mitomycin, followed by measurement and comparison of T cell proliferation of un-transfection group, empty group and transfection group with MTT assay.(6)After its inhibition with mitomycin, sensitized DC were co-cultured with homogenous T cells and parental H640 cells at certain ratio for 72h, followed by measurement and comparison of inhibition on proliferation of parental H460 cells of un-transfection group , empty group and transfection group with MTT assay.Results:1.CD40L gene could be obtained by reverse transcription of total RNA extracted from activated human T cells and following PCR Amplification.2.DNA sequencing of insertion fragment was performed after the screening of recombinant pMD-18-T-CD40L, and the acquired data showed that it fully matched the 40 to 915 base sequence of BC071754 CD40L cited by NCBI nucleic acid database.3.Recombinant expression vector pcDNA3.1-CD40L was obtained after digestion and recombination of pMD-18-T-CD40L and eukaryotic expression vector pcDNA3.1(+)plasmid, identification of inserted DNA sequence in this plasmid was performed by enzyme digestion ,PCR, and DNA sequencing. The results showed that it fully matched the 40 to 915 base sequence of BC071754 CD40L cited by NCBI nucleic acid database with right direction.4.PCR detection showed that there was CD40L mRNA transcription in H446 cells transfected by pcDNA3.1-CD40L.The antibodies of CD40L marked by PE were tested by flow cytometry, and there was CD40L in transfected cells. It showed the recombinant vector could play a normal role in translation of CD40L, transcription and protein synthesis.5.There were differences among the expressions systems of different configurations of pcDNA3.1-CD40L, so were the expression of different shapes of transfected cells. Expression of circular plasmid was very high in H446 cells, while expression of linear plasmid was higher in SK-hepⅠcells with statistical significance.6.pcDNA3.1-CD40L was transfected into three cell lines: SK-hepⅠ,H446 and H460 with significant difference of expression rate, which was H446> SK-hepⅠ> H460 in order. 7. CD40L was expressed in both empty vector group and transfection group after transfection of pcDNA3.1-CD40L and empty vector into primary pulmonary carcinoma cells, but the expression was much higher in the transfection group than empty vector group with statistical significance.8.Expression of CD40L in recombinant plasmid H460 could reach more than 80% after G418 screening.9.After they were sensitized by prepared H460 cells expressing CD40L, DC cells had higher expression of surface molecules such as CD83,MHC-Ⅱcompared with that in empty vector group with statistical significance. The expression of CD86 was higher than that in empty vector group, but there had not been any statistical significance so far.10.Compared to un-transfected group and empty vector group, DC in CD40L transfected group had higher secretory volume of LI-12, greater ability in mediating the multiplication of homogeneous T cells and the inhibition on proliferation of parental H460 cells after they were sensitized by sensitizers with statistical significance.Conclusion:1.We successfully constructed the human CD40L expression vector, which laid foundation for further clinical researches of anti-tumor vaccines with CD40L.2.Human CD40 expression vectors were transfected with different configurations in different cells, and their expression rates showed significant differences, which was relevant to different physiological states of cells.3.The effective expression of CD40L expression vector in primary pulmonary carcinoma cells laid foundation for further clinical researches.4.Compared to DC sensitized by tumor cells with no expression of CD40L, DC had obvious higher expression rate of costimulatory molecules and greater secretory volume of IL-12, greater ability in mediating the multiplication of homogenous T cells as well as the growth inhibition on homogenous tumor cells when they were sensitized by prepared tumor cells with expression of transfected CD40L.