Establishment Of T Lymphocytes Secreting EphrinAl-Caspase-3

Background: Among all kinds of high-grade female malignant tumors,the incidence of breast cancer is getting higher and higher,and it ranks first among malignant tumor deaths.Therefore,more molecular biomarkers and new targets for treatment need to be discovered for diagnosis and prognosis.To develop new molecularly targeted drugs.Eph receptors are the largest receptor tyrosine kinase(RTKs)subfamily and are an important part of the cellular signaling pathway.The EphA2 receptor is a member of the Eph receptor,and its specific ligand is EphrinA1.In recent years,more and more researchers have begun to pay attention to the role of EphA2 receptor and its ligand EphrinA1 in breast cancer.EphA2 receptor and its ligand EphrinA1 will become a new target and new in the diagnosis and treatment of breast cancer.Molecular markers.Caspsae-3 is an important effector molecule for performing apoptosis,and most of the apoptotic pathways are caused by Caspase-3.Radiation therapy and chemotherapy for treating tumors depend on apoptosis leading to cancer cell death,so it can be used as an enhancer for anti-tumor drugs to induce apoptosis in tumor therapy,and provide a new idea in the future treatment of tumors.Objective: In this study,T lymphocytes were cultured,and T lymphocytes were infected by the constructed Ad-Ephrin Al-Caspase-3 recombinant adenovirus to investigate whether it can secrete the apoptosis-promoting gene EphrinAl-Caspase-3after infection,and in which EphrinAl and The expression of Caspase-3.Methods:1.Cultured T lymphocytes2.T lymphocytes were infected by recombinant adenovirus Ad-Ephrin Al-Caspase-3 with different multiplicity of infection(MOI),and the amount of cells expressing EphrinAl was detected by immunofluorescence.3.The proliferation and toxicity of recombinant adenovirus Ad-Ephrin Al-Caspase-3 infected T lymphocytes were detected by MTT assay.4.The expression of EphrinAl-Caspase-3 in T lymphocytes after infection was detected by quantitative PCR assay and ELISA assay.Result:1.The cultured T lymphocytes are regular in shape and uniform in size.2.The amount of cells expressing EphrinAl was detected by indirect immunofluorescence.As the MOI increased,the cells expressing EphrinAl increased(green fluorescence).When the MOI was 100,the number of cells expressing EphrinAl was the highest,and then increased with the increase of MOI(200).EphrinAl has a reduced number of cells,and a high MOI virus may affect cell survival.3.Cell proliferation and toxicity were measured using the MTT assay,and cell proliferation activity decreased with increasing MOI.As time goes by,the inhibitory effect of the virus on cell growth is becoming more and more obvious.4.The expression of EphrinAl-Caspase-3 was detected by quantitative PCR and ELISA,and the expression of EphrinAl and Caspase3 was increased.Conclusion: The expression of EphrinAl and Caspase-3 was increased after infection of T lymphocytes by Ad-EphrinAl-Caspase-3 recombinant adenovirus.With the increase of MOI,the number of cells decreased and the cell proliferation activity decreased,and it was time-and dose-dependent.For the follow-up study,the in vitro and in vivo targeting of the proapoptotic gene EphrinAl-Caspase-3 lays a foundation for the killing effect of breast cancer cells.