| Background&Objective As a digestive tract malignant tumor, esophageal cancer is a serious threat to human health. Its morbidity and mortality has been very high for many years, China is one of the high incidence areas. In recent years, with the development of cognitive level, people have done a large number of clinical and basic studies and made considerable progress in the occurrence and development, diagnosis and therapy of esophageal cancer. However, the prevention and control work has not yet reached a satisfactory level, the5year survival rate of esophageal cancer is always less than30%. Tumor progression is a process of multiple factors, genes and stages. As a special type of T cell subgroups. CD4+CD25+Foxp3+regulatory T cells have played an important role in the development of cancer. They could promote the tumor cells excaping from the body’s immune response, and the expression of them was up-regulated in gastric cancer, lung cancer, breast cancer, ovarian cancer and other malignant tumors. How to reduce their inhibitory effect on tumor immune response and achieve desired purpose of immune treatment has become a research hot spot. However, the research about downregulation of regulatory T cells and esophageal tumor immunity has been rarely reported at home and abroad. The aim of this study is to observe the regulatory T cells downregulation and its impact on antitumor immune effect in mice by CD25, Foxp3monoclonal antibody tail vein injection to mice bearing human esophageal cancer, and then provide basis for the immuno-biological therapy of esophageal cancer.Methods Transplanted the human esophageal tumor tissues to the abdominal cavity of48BALB/c mice, and then divided them into4groups:control group, CD25monoclonal antibody group, Foxp3monoclonal antibody group and combined monoclonal antibody group. Each mouse of control group was injected with0.2ml PBS through tail vein, while the mice of CD25monoclonal antibody group, Foxp3monoclonal antibody group and combined monoclonal antibody group received each injection of125ug CD25monoclonal antibody,50ug Foxp3monoclonal antibody,62.5ug CD25monoclonal antibody combined with25ug Foxp3monoclonal antibody respectively. The injection was carried3days before and1day after the transplantion.2and4days after transplantation,2mice in each group were killed to observe the tumor growth and decline, lymphocyte infiltration.6days after transplantation, the remain mice were killed to observe the general condition, tumor growth and decline, lymphocyte infiltration, immune organ index, Treg cells expression in peripheral blood and Foxp3immunohistochemical staining intensity of transplanted tumor. Then analyzed the downregulation of regulatory T cells and its impact on antitumor immune effect and pathological changes of transplanted tumor tissue. Input all data to SPSS15.0software to establish a database and analysis. ANOVA analysis was used to compare the mice weight before transplantion and after being killed, transplanted tumor initial weight and volume, thymus and spleen index and expression of regulatory T cells among groups. Kruskal-Wallis test was used to compare the transplanted tumor weight and volume after the mice were killed. Fisher’s exact probability and pairwise comparison of multiple sample rate were used to compare lymphocyte infiltration and Foxp3immunohistochemical staining intensity of transplanted tumor among groups. P<0.05represented a statistically significant difference, when comparing pairwise sample multiple sample rate, adjust the inspection level according to the formula of α’=α/[k(k-1)], P<α’represented a statistically significant difference. Results1. Before death, the general condition of all mice was well, there were no statistical differences of the mice weight before and after transplantion among groups (P>0.05).2. The proportions of regulatory T cells in CD4+T cells in groups were (7.083±2.002)%,(1.934±1.186)%,(5.738±1.772)%and (3.772±1.120)%respectively. Proportions of CD25monoclonal antibody group and combined monoclonal antibody group were lower than other two groups. There were statistical differences of proportion among groups (P<0.05).3. There were no statistical differences of transplanted tumor initial weight and volume among groups (P>0.05). The tumor tissues all had different degrees of necrosis, and the weight and volume all decreased compared with those before transplantion. The transplanting tumor weight and volume in antibody injection groups were less than those of control group. The tumor cells in CD25monoclonal antibody group were rejected soon. The visibility of the tumor cells decreased significantly6days after the transplantion, the tumor weight and volume was ’minimal. And then followed by combined monoclonal antibody group and Foxp3monoclonal antibody group.4. The thymus indexs of the groups were (17.483±4.998),(35.234±11.354),(21.441±4.754) and (28.512±7.015)10mg.g-1respectively. And the spleen indexs of the groups were (32.547±10.824),(47.083±12.133).(34.618±12.153) and (41.897±18.905)10mg.g-1respectively. There were statistical differences of immune organ inde among groups (P<0.05). The immune organ indexs of CD25and combined monoclonal antibody group were significantly higher than the control group (P<0.01), while those in Foxp3monoclonal antibody group had no statistically significant difference compared with the control group. Compared with CD25monoclonal antibody group, the immune organ indexs of Foxp3and combined monoclonal antibody group were significantly lower (Foxp3&CD25monoclonal antibody group P<0.01, combined&CD25monoclonal antibody group P<0.05). Compared with Foxp3monoclonal antibody group, the immune organ indexs of combined monoclonal antibody group were significantly higher (P<0.05). 5. There were statistical differences of inflammatory cell infiltration degree among groups (P<0.05). The inflammatory cell infiltration degree of CD25and combined monoclonal antibody group were higher than those of the control group. While those in Foxp3and combined monoclonal antibody group were lower than CD25monoclonal antibody group. The inflammatory cell infiltration degree of combined monoclonal antibody group was lower than the Foxp3monoclonal antibody group. The tumor of CD25and combined monoclonal antibody group appeared lymphocytic infiltration in the early stage. As time migrating, the tumor cells degenerated and disappeared gradually, lymphocyte infiltration and fibrous hyperplasia gradually increased. The tumor cells atypia of control and Foxp3monoclonal antibody group was still very high, the time of lymphocytic infiltration and tumor necrocytosis was later than the above two groups, and the degree of fibrous hyperplasia and lymphocyte infiltration were lower than the above two groups.6. The Foxp3immumohistochemical staining intensity of the patient’s esophageal cancer tissue was strong positive (+++). Foxp3mainly expressed in the nuclei and cytoplasm. The Foxp3expression all decreased after the transplantion, there were statistical differences of Foxp3expression among groups (P<0.05). In control group, the positive expression rate was87.5%. in which6cases were weakly positive (+), lease was moderate positive (++). In Foxp3monoclonal antibody group.6cases were weakly positive (75%), the other2cases were negative. In combined monoclonal antibody group, the positive and negative cases accounted for50%respectively. The Foxp3expression positive rate of CD25monoclonal antibody group was lowest, and then followed by combined, Foxp3monoclonal antibody and control group. However, when compared respectively, there was no significant statistical difference between the control and Foxp3monoclonal antibody group. CD25and combined monoclonal antibody group.Conclusions In CD25monoclonal antibody group, the expression of regulatory T cells was lowest, the transplantation tumor necrosis degree was highest and the reduction of transplantation tumor weight and volume was the most. And then followed by combined monoclonal antibody group. There was no statistical difference of regulatory T cells expression between Foxp3and combined monoclonal antibody group. The reduction of transplantation tumor weight and volume of them were also less than the above two groups. Regulatory T cells plays a vital role in esophageal cancer occurrence and development, and their negative immune inhibition mechanism remains to be further explored. CD25monoclonal antibody could downregulate regulatory T cells effectively in mice, and then reduce the immunosuppressive function of the cells and enhance the antitumor immune effect while Foxp3monoclonal antibody was hard to have effect on Foxp3nuclear expression. Immune therapy targeted Foxp3molecule still needs further study. In general, as an diagnosis molecular marker and clinical new targets of esophageal cancer, immunotherapy against regulatory T cells still needs better improvement, so as to provide more effective clinical treatments for esophageal cancer. |
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