The Role Of CagA、MACC1 And Th17 Cells In The Development Of Gastric Cancer

Object:Helicobacter pylori infection is one of the main causes of gastric cancer, we firstly investigated the antibody to virulence protein Cag A of Helicobacter pylori in the serum of gastric cancer patients and then analyszed the expression of MACC1 in gastric cancer patients, the IL-17A+/IFN-γ+CD3+T(CD4、CD8、γδT) and related cytokines(IL-17A、IL-23、IL-1β、IFN-γ) and the transcription factor(RORγt、T-bet) expression level of patients with gastric cancer, and explored: IL-17A+/IFN-γ+CD3+T cells and factor after preoperative/changes; expression in peripheral blood and tissue;observe its relationship with the clinical histopathological classification, and preliminary discuss the possible role.Methods:1) The peripheral blood specimens of patients with gastric cancer and healthy cases of sex and age matched with gastric cancer patients were all from the second hospital affiliated to Suzhou university; all patients specimen are recorded the gender, age, pathological tissue TNM classification data;2) Constract the prokaryotic expression vector of Helicobacter pylori Cag A protein. The Cag A protein was prepared and used for the detection of the anti Cag A antibodies in the gastric cancer patients serum by ELISA;3) The expression of MACC1 in the tissues of gastric cancer patients and adjacent tissues was detected by RT-PCR. At the same time, the MACC1 expression in gastric cancer tissues was investigated by the immunohistochemical technique and correlation with clinical pathological parameters was analyzed.4) Separate peripheral blood mononuclear cell(PBMC) of the healthy cases and gastric cancer patients,(1 x 106 / cells), and stimulation of 5 with PMA(ng/ml) of 50, Ionomycin mu(1 g/ml) and Monensin(500 ng/ml); Detect frequency of IL-17A+/IFN-γ+CD3+T(CD4 、 CD8 、 γδT) cells by multicolor flow cytometry instrument(BD FACSVerse flow cytometer).5) With real-time fluorescence quantitative polymerase chain reaction(QRT- PCR) technology, we detected the IL-17A+T cells transcription factor RORγt,IFN-γ+T cells related transcription factors T-bet and IL-17A+/IFN-γ+T cells related cytokines IL-17A、IL-23、IL-1β、IFN-γ m RNA expression level in peripheral blood PBMC and tumor tissue.6) Using liquid chip technology and enzyme-linked immunosorbent assay(ELISA) method to detect serum IL-17A、IL-23、IL-1β、IFN-γ concentration in patients with gastric cancer and healthy cases.7) Using SPSS13.0 software to analysis the expression of MACC1 in the tissues of gastric cancer patients and adjacent tissues、 the frequency of IL-17A+/IFN-γ+CD3+T cells and the related cytokines levels, pathological staging gastric cancer data.Results:1) The high purity of Cag A fusion protein was obtained. The positive rate of antibody for Cag A in the gastric cancer patients’ serum is as high as 83.3% and its relation with the patient’s age, gender, and tumor infiltration depth, lymph node metastasis and clinical pathologic stage etc was not observed. While the positive rate of antibody for Cag A in healthy control is only 5%.2) The positive rate of MACC1 m RNA in gastric cancer tissues was 70%, significantly higher than that of adjacent tissue. The expression of MACC1 m RNA and gastric cancer infiltration depth, TNM stages, differentiation degree and the presence of lymph node metastasis were positively correlated, but its relation with age, sex and tumor size was not observed. The immunohistochemical experiments results showed that the expression of MACC1 protein in gastric cancer tissues is high, and the expression of MACC1 protein in the corresponding adjacent tissue can not be detectd.3) The frequencies of circulating Th17 and γδT17 cells in CD3 + T cells were significantly higher in patients with GC preoperatively than in HDs, while Tc17 cell frequency decreased significantly in the CD3 + T cells. Additionally, the frequency of Th17 cells was negatively associated with that of Tc17 cells in patients with GC. The percentage of IFN-γ+CD3+CD4+Th1 cells, IFN-γ+CD3+CD8+T cells and IFN-γ+CD3+γδT cells in peripheral blood in patients with gastric cancer is significantly lower than healthy group.2) There are significant differences among TTh17、Tc17、IFN-γ+CD3+CD4+T、IFN-γ+CD3+CD8+Tcells in the peripheral blood and tumor infiltration depth(T1+T2 / T3+T4) and tumor distant metastasis(M0 / M1) respectively; there are significant differences in Tc17、γδT17、IFN-γ+CD3+CD8+T 及 IFN-γ+CD3+γδT cells in the TNM staging(I + II/III, IV); there are significant differences among γδT17、IFN-γ+CD3+γδT cells lymph node close and metastasis of tumor(N0 / N1 + N2 + N3).4) The main source of cell factor IL- 17 in CD3 + T cells in peripheral blood is Th17 cells, followed by Tc17 cells, and the last is γδT17 cells; Within the cells, the main sources of IFN-γ+ cell factor in CD3 + T cells in peripheral blood is CD8 + T cells, followed by CD4 + Th1 cells, and the last proportion is γδT cells.5) The serum of IL-17A、IL-23、IL-1β concentration in gastric cancer patients are significantly higher than that of healthy group, and the concentration of IFN-gamma was significantly lower than healthy group; there are significant differences between IL-17A、IL-23、IL-1β and IFN-γ in cancer lymph node close transfer(N0 / N1 + N2 + N3) and TNM staging(I + II/III, IV); there are significant differences in IL-23、IL-1β、IFN-γ density in tumor infiltration depth(T1 + T2 / T3,+T4) respectively; IL-1β in tumor differentiation degree( Well/Moderate vs. Poor) also exist certain differences; Additionally, serum IL- 17 A concentration is closely related to the frequency of Th17 cells in gastric cancer patients(r = 0.3310, p = 0.3310).6) The expression of transcription factor of IL-17A+CD3+T cells, RORγt m RNA, in gastric cancer patients were significantly higher than that of healthy cases, but the expression of IIFN-γ+CD3+T of transcription factor, T-bet m RNA, was significantly lower than healthy cases(P < 0.05).Conclusion:1) In China, the development of gastric cancer was mostly associated with the Cag A positive Helicobacter pylori infection. Cag A antibody is expected to become a new high risk factor of gastric cancer.2) The expression of and infiltrating depth of gastric cancer, TNM stage, differentiation degree and the presence of lymph node metastasis were positively correlated. MACC1 is expected to become a new biomarker for the diagnosis and treatment of gastric cancer.3) In patients with gastric cancer, Th17 and γδT17 has high expression in CD3+T cells in peripheral blood, while Tc17 cells show lower expression, and the main sources of intracellular IL- 17 A is Th17 cells;4) In patients with gastric cancer, IFN-γ+CD3+CD4+Th1、IFN-γ+CD3+CD8+T and IFN-γ+CD3+γδTcells show lower expression in peripheral blood CD3 + T cells, and the main sources of intracellular IFN-γ is CD3+CD8+T cells;5) The serum concentration and the m RNA expression level of IL-17A、IL-23、IL-1β and m RNA expression level of transcription factors(RORγt) were significantly higher than that of healthy cases, Serum concentration of IFN-γ, were lower than that in healthy cases, which is related with tumor pathological features.