Expression Of Costimulatory Molecule B7-H1 In Laryngeal Squamous Cell Carcinoma-associated Fibroblasts And Its Immune Function

Objectives: Observe the expression of B7-H1 on fibroblasts in tumor tissue of patients with laryngeal squamous cell carcinoma.To explore the effect of B7-H1 on tumor cell proliferation,migration and immune evasion in the tumor microenvironment by regulating the expression of B7-H1 in CAFs.This study provides a theoretical basis for the application of immunotherapy to regulate T cells in LSCC.In order to reduce the head and neck organs and their functional damage caused by traditional treatment,new ideas are provided.Methods:Twenty patients with confirmed laryngeal squamous cell carcinoma were enrolled in this study.The specimens were taken from the normal margin of the larynx.Frozen sections were taken and immunofluorescently stained.B7-H1 expression was observed under a microscope.The larval squamous cell carcinoma tumor-associated fibroblasts(CAFs)and normal fibroblasts(NFs)were isolated and purified from the specimens,purified and identified by fluorescence.The expression of B7-H1 in purified CAFs and NFs was detected by flow cytometry.According to the results,transfection with si RNA down-regulated the expression of B7-H1 in CAFs and detected the expression of B7-H1 at the m RNA level by RT-PCR.The differences and changes of CAFs between CAFs and B7H1-si RNA transfection groups were compared by proliferation experiments and scratch experiments.Co-culture experiments were performed to compare the differences in the co-culture of CAFs and HEP-2 cells under different B7-H1 expression conditions.The co-culture of CAFs and T cells in different B7-H1 expression conditions was also compared.And verified by multiple cytokine assays.Results:(1)B7-H1 expression was found in 15 specimens of patients with laryngeal squamous cell carcinoma,but no B7-H1 expression was observed in the normal group.After 3 generations of CAFs culture,NFs were purified after 2 passages of culture.CAFs and NFs were positive for vimentin,while CAFs were positive for ?-smooth muscle actin(?-SMA,CAFs specific protein).(2)The flow-through results showed that the expression of B7-H1 on the surface of CAFs was significantly higher than that of NFs(P<0.05).(3)RT-PCR results showed that B7-H1 was significantly lower in the B7H1-si RNA transfection group than in the control-si RNA group.(4)The results of cell proliferation experiments showed that the proliferation ability of B7H1-si RNA transfection group was significantly lower than that of untreated group(P<0.05).The scratch test showed that the migration ability of B7H1-si RNA transfection group was lower than that of untreated group(P< 0.05).(5)Hep-2 cell co-culture experiments showed that the proliferation of tumor cells in the B7H1-si RNA transfection group was significantly lower than that in the untreated group(P<0.05).T cell co-culture experiments and ELISA assays showed that the proliferation of T cells in the B7H1-si RNA transfection group was significantly higher than that in the untreated group(P<0.05);compared with the untreated group,B7H1-si RNA transfer IL-2 secretion increased and IL-10 secretion decreased in the infected group.Conclusions:The expression of B7-H1 in CAFs of patients with LSCC was higher than that of NFs.Decreasing the expression of B7-H1 on CAFs could significantly reduce the proliferation and migration of CAFs.And reduce the proliferation of Hep-2 cells,enhance the proliferation and activation of T cells,and enhance the immune function of T cells.