The Study On The Anti-Cervical Cancer Immunity Induced By Dendritic Cells Derived From Patients Monocytes In Vitro

Objective To study the methods of induction, biological character of dendritic cells(DCs) which derived from the peripheral blood of patients with cervical cancer, and further to study the anti-tumor effect activated by DCs in vitro. It will provide the experimental foundation for clinical immunotherapy of DCs.Methods1．The induction and biological character of DCs derived from human monocytes Monocytes isolated from the peripheral blood of patients with cervical cancer are cultured at 37℃ in humidified 5% CO2 in air in medium with rhGM-CSF,rhIL-4 and TNF-α for 7-10 days. DCs from human monocytes are analyzed on morphology, cellar structure under optics and electronic microscope; and then phenotype of DCs are detected by FACS analysis including specific CD1α, and function-related molecules HLA-DR, CD80. Stronger allo-MLR is one of DCs' characteristics, it is performed by using MTT method to observe the ability of DCs stimulating lymphocytes proliferation.2．The ability of DCs loaded with tumor cell antigen stimulating lymphocytes proliferation In order to study the ability of DCs which loaded with antigen stimulating lymphocytes proliferation, monocytes isolated from the peripheral blood of patients with cervical cancer are cultured in medium with rhGM-CSF, rhIL-4 and TNF-α. At the same time, got the tumor cell antigen from cervical tumor tissue of the same patient. Gain T lymphocytes by using cytokine IL-2 to stimulate antologous lymphocytes. Then cultured the tumor antigen, DCs and T lymphocytes all together, the proliferation of T lymphocytes in the different days are detected respectively by using MTT method.3．DCs and anti-cervical cancer immunity Autologous lymphocytes proliferation activated by induced DCs loaded with tumor cell antigen is detected by using MTT method to observe the presenting role of DCs. To detect the specific cytotoxic effect activated by DCs on Hela and SKOV3 cells, CTL activity is observed by counting the killed Hela and SKOV3 cells in vitro. ResultThe identification of DCs and its biological character Typical DCs can be induced when monocytes are cultured in medium with rhM-CSF,rhIL-4 and TNF-α for 7 days. It shows typical morphology and cellular structure of dendritic cell under optics and electronic microscope. It highly express the specific cellular phenotype CD1α and other APC-related functional molecules CD80 and HLA-DR. The detection of allo-MLR shows that DCs can stimulate strong T lymphocytes proliferation.The ability of DCs loaded with tumor cell antigen stimulating lymphocytes proliferation The optical densities of 24h,48h,72h,96h are detected respectively, the stimulating indexs show that DCs loaded with tumor cell antigen can stimulating strong antologous lymphocyte proliferation, it is more effective than control's(P<0.05).DCs anti-cervical cancer immunity DCs loaded with tumor cell antigen can generate strong specific CTL that kill the target cells—Hela cells(56.410%), SKOV3 cells(24.901%), while CTLs induced by DCs which cultured without antigen kill the target cells weakly(10.256% and 20.151% respectively).T lymphocytes stimulated by tumor antigen only generate non-specific immunoreaction, kill rates are 1.865% and 15.613%. ConclusionCulturing human monocytes in medium with rhGM-CSF,rhIL-4 and TNF-α can induce typical DCs. It shows potent antigen presenting role to exogenous antigen.DCs from the peripheral blood of patients with cervical cancer pulsed by tumor cell antigen not only strongly stimulate autologous lymphocytes proliferation, but also play an important role in anti-tumor immunity.DCs loaded with tumor cell antigen derived from patient monocytes can generate specific CTLs that have potent cytotoxic effect on cervical cancer Hela target cells. According to yhe experimental result, we put forward the theory on the treatment of cervical cancer by the use of DCs vaccine.