Study On The Therapy Of Cervical Cancer In Mice Model By Semi-allogeneic Effector T Lymphocytes

Objective: The tumor-carrying mice model was setted up by subcuta-neously injecting the U14cells into the C57BL/6mouse. Targeting thetransplantable cervical cancer in C57BL/6mice, the effector T lymphocyteswere derived from the C57BL/6female mice and the CB6F1mice（BABL/C×C57BL/6hybrid generation, female）. This study observed theexpressions of STAT3, P-STAT3, TGF-β₁, and IFN-γ in the tumor tissue, thesurvival period, and the level of the graft anti-host reaction （GVHR） indifferent groups; and to explore the anti-tumor efficacy of the autologous andsemi-allogeneic effector T lymphocytes. This study will provide new ideas forclinical treatment of cervical cancer.Methods：1The cervical carcinoma U14cells were cultured in vitro. The cells wereused to set up the U14-carrying mice model.2Preparation of tumor lysate-pulsed dendritic cells: They were obtainedby culturing the C57BL/6or CB6F1mice bone marrow cells, then adding thermGM-CSF, rmIL-4, tumor cells lysate, and LPS into the medium. Theexpressions of the CD11c⁺, MHCII⁺and CD86⁺cells’ surface molecules wereanalyzed by the flow cytometry.3Preparation of effector T lymphocytes: The C57BL/6, CB6F1micespleen lymphocytes were separated by the lymphocyte separating medium,and then we could obtain the T lymphocytes by the nylon column. Tlymphocytes were Cultured for2days, and the tumor lysate-pulsed dendriticcells were added to form the effector T lymphocytes.4The cytotoxicity assay in vitro: The C57BL/6, CB6F1effector Tlymphocytes and mice cervical carcinoma U14cells were mixed by50:1and25:1, and cultured for8hours. The lactate dehydrogenase concentration in the supernatant of the medium was analyzed by the lactate dehydrogenase kit, andcalculated to get the killing rates.5The treatment in different groups: The U14-carrying mice were dividedinto3groups with8mice in each group. Control group （A group）: PBS wasinjected by vein for0.5ml, every5days once, repeated3times. Autologouseffector T cells group （B group）: effector T cells of C57BL/6mice wereinjected by vein for1×10⁶per0.5ml, every5days once, repeated3times.Semi-allogeneic effector T cells group （C group）: effector T cells of CB6F1mice were injected by vein for1×10⁶per0.5ml, every five days once, repeated3times. Calculated the survival period of different groups.6Immunohistochemistry method was used to detect the expression ofSTAT3, P-STAT3, TGF-β₁, and IFN-gamma of tumor tissue in differentgroups, their content were expressed by the percentage of the positive cells,which were analyzed by the image analysis software.7Observing tumor tissue and pathological changes of liver, smallintestine in mice in HE staining.Results：1The time of setting up U14-carrying mice model was about7days.2The mature dendritic cells were suspending, having dendritic prominen-cies, the purity of the cell surface molecules CD11c⁺MHC II⁺was92.4%，and the CD11c⁺CD86⁺was88.8%.3Cytotoxicity of effector T cells in vitro: when effector-target ratio was50:1, the killing rates between the C57BL/6and CB6F1effector lymphocyteshad no statistically significant differences （P>0.05）. When that ratio was25:1,the killing rates between the C57BL/6and CB6F1lymphocytes also had nostatistically significant differences （P>0.05）.4The survival period of different groups: A and B groups, A and Cgroups, both had statistical difference （P<0.05）. B and C groups had nostatistically significant difference （P>0.05）.5The results of immunohistochemistry: Expressions of TGF-β₁, IFN-γSTAT3, and P-STAT3between A group and C group had statistical difference （P<0.05）. Expressions of TGF-β₁and P-STAT3between A group and B grouphad statistical difference （P<0.05）, IFN-γ and STAT3between A group and Bgroup had no statistically significant difference （P>0.05）. Expressions ofTGF-β₁, P-STAT3and IFN-γ between B group and C group had statisticaldifference（P<0.05）, STAT3between B group and C group had no statisticaldifference （P>0.05）.6HE staining results: Observing the tumor tissues of mice, B group andC group had more necrosis areas than A group. Liver and small intestine ofdifferent groups had no obvious pathological changes.Conclusion：1C57BL/6, CB6F1effector T cells have antitumor activity in vitro. Boththe killing rates have no statistical difference.2C57BL/6effector T cells can break the immunologic suppression,inhibit the expression of the TGF-β₁, and decrease the P-STAT3.3CB6F1effector T cells can decrease the expressions of the STAT3, theP-STAT3, and the TGF-β₁, and promote the expression of the IFN-γ, andenhance more effective antitumor immune response.4There was no obvious graft anti-host reaction in tumor-carrying mice,which had been injected with C57BL/6, CB6F1effector T cells.5C57BL/6, CB6F1effector T cells can prolong the survival period of thetumor-carrying C57BL/6mice. The periods have no statistical difference.