Study On The Preparation And Anti-Tumor Biological Effects Of Dendritic Cell-Renal Cell Carcinoma 786-O Cell Line Fusion Vaccines In Vitro

Objective:To prepare and select pure Dendritic cell-Renal cell carcinoma 786-O cell line fusion vaccine(DC-RCC786-O),By researching its biological characters and anti-tumor effects in vitro, we hoped to provide the theory foundation and experimental basis for renal cell carcinoma's immunotherapy which based on Dendritic cells.Methods:1.Prepare and observe the morphology characteristics of DC-RCC786-O fusion vaccines. Peripheral blood mononuclear cells were separated from healthy adults'peripheral blood by density gradient centrifugation,co-cultured with recombinant human Granulocyte-Macrophage colony stimulating factors(rhGM-CSF),recombinant human Interleukin-4(rhIL-4)and Tumor necrosis factor-α(TNF-α)for 7 to 9 days to generate mature DC,Examine the expression of cell surface phenotypes by flow cytometry.The frozen stored RCC786-O cell lines were resuscitated and cultured,RCC fused with DC at a ratio of 5:1 by using of polyethylene glycol(PEG).Discard the non-adherent cells after 24 hours'culture, the adherent cells co-cultured with the same cytokine for another 4 days, and the pure fusion cells could be screened out by the adherent incubating method;Observe the morphology characteristics through light microscope and electron microscope. 2.Biological characters and anti-tumor effects of DC-RCC786-O fusion vaccines in vitro. Describe the cell growth curves;The ability to amplify allogenic T lymphocytes was assessed through mixed lymphocyte reaction(MLR);Examine the expression of cell surface phenotypes(CD86,HLA-DR) by flow cytometry(FCM);Detect whether the hypodermic injection of fusion vaccine could proliferate to be a new carcinoma in severe combined immunodeficient(SCID)mice.The ability to irritate cytotoxic T lymphocytes(CTL) to induce anti-tumor immune reaction in vitro assessed through MTT method and the effect to target cell's divide cycles was detected by FCM.Results:1.We obtained mature DC from peripheral blood mononuclear cells by co-cultured with rhMG-CSF, rhIL-4 and TNF-αfor 7 to 9 days. The mature DC had typical morphological characteristics and the cell phenotypes were CD86(62.07±2.01) %, HLA-DR(66.30±1.51) % respectively;DC and RCC786-O can be fused effectively by PEG and we could get pure fusion cells by using adherent culture method.Fusion cell's volume was much bigger than DC or RCC, it lived in suspension in the culture medium and could not proliferate to be new carcinomas in SCID mice.2.DC-RCC786-O fusion cells could divide and proliferate in vitro,but their growth curve is more flat than that of RCC,fusion cells could not proliferate to be new carcinomas in SCID mice;The fusion cell phenotypes were CD86(81.23±1.01)%,HLA-DR(80.16±1.11)%respectively,which was extensively higher than that of DC control group (P<0.05);Fusion cells also could strongly irritate the proliferation of T lymphocytes,which was extensively higher than that of DC control group and mixed cultured DC group(P<0.05),A peak response of MLR will be achieved at stimulator to responder ratio of 1:1; Fusion cells have the strongest ability to irritate cytotoxic T lymphocytes to induce anti-tumor immune reaction compared to the control group in vitro,MTT assay showed the inhibition ratio is (75.49±5.10)%,which is significantly higher than that of DC control group (45.44±2.87)%(P<0.05)and the mixed cultured DC group(14.55±0.75)% (P<0.05).Conclusion:1.Peripheral blood mononuclear cell co-cultured with rhGM-CSF, rhIL-4 and TNF-αcan obtain mature DC,which has its unique morphous and phenotypes; DC and RCC can be fused effectively by PEG, We can obtain pure DC-RCC786-O fusion cell by using adherent culture method.2.DC-RCC786-O fusion cell lives in suspension and can divide and proliferate in the culture medium, but its growth ability is decreased significantly as compared with RCC and can not proliferate to be new carcinomas in SCID mice. The fusion cell's surface phenotypes are remarkably increased.The fusion cell has distinguished effect on proliferating T lymphocytes and inducing the effector cell CTL to inhibit and kill the target RCC786-O cell in vitro.3.DC-RCC786-O fusion cell which based on DC can provide a new and effective method to RCC's immunoltherapy.