| ?Background?Gastric cancer is the fifth common cancer in the world.It is the second most lethal cancer in the world,and more than 60%of gastric cancer cases are distributed in developing countries.The total survival rate of patients with gastric cancer is better than before,but the 5-year survival rate is still lower than 30%,which can seriously harm people's health.The inflammatory response of the body to tumor cells may affect the progress of tumor.Systemic inflammatory response markers such as neutrophil/lymphocyte ratio(NLR)are being studied to determine whether they can affect the prognosis of patients with malignant tumor.Neutrophils not only have the function of phagocytosis,sterilization and activation of the immune system,but also exist in tumor and adjacent tissues.These infiltrated neutrophils are called tumor associated neutrophils(TANs).TANs are relevant to tumor growth,metastasis and angiogenesis.Under the action of different cytokines in tumor microcirculation,tans can polarize to N1 and N2phenotypes and play different functions.Met is a transmembrane tyrosine kinase receptor encoded by gene met.Met is not only expressed on the surface of tumor cells,but also in immune cells.It was found that Met expressed on the surface of neutrophils plays an important role in inhibiting tumor progression.This study will explore whether Met~+neutrophils can inhibit the growth,invasion,metastasis and other malignant behaviors of gastric cancer,and the role of MET gene in the polarization of TANs subpopulation.At the same time,we will study the influence of peripheral blood NLR on the clinical characteristics,such as tumor stage,differentiation and so on and prognosis of gastric cancer patients.It is significant for us to judge the progress of tumor patients and make targeted treatment plans.?Methods?1.The effect of Met~+neutrophils on the growth and metastasis of gastric cancer The peripheral blood neutrophils were extracted by Ficoll Hypaque density gradient centrifugation and Met~+neutrophils were screened by FACS.With Met~+neutrophils as the experimental group and Met~-neutrophils as the control group,we co-cultured with gastric cancer cell lines to detect the proliferation,cell viability,apoptosis,invasion,migration and other functional changes of gastric cancer cells.2.The role of HGF/Met signaling pathway in TANs polarizationSelect Met~+neutrophils as the experimental group,and set up blank control group,gastric cancer cell culture supernatant group,HGF group,anti met group,gastric cancer cell culture supernatant+Anti-met group,HGF+Anti-met culture group,select Met~-neutrophils as the control group.The expression of ICAM-1,Fas,TNF-?,CASP3 and i NOS were detected by WB and PCR.The killing ability of neutrophils in each group was tested by flow cytometry.In96 well plate,neutrophils and labeled gastric cancer cells were added according to the proportion of neutrophils/gastric cancer cells in each treatment group.After incubation,they were centrifuged,the supernatant was extracted and tested by computer.3.Relationship between neutrophil/lymphocyte ratio and prognosis in patients with gastric cancer undergoing chemotherapyWe divided the gastric cancer patients who received chemotherapy into high NLR group and low NLR group,and counted the clinical characteristics of tumor stage,tumor pathological differentiation,total survival time,disease-free survival time and so on.The results of clinical correlation analysis showed that the increase of inflammatory factor neutrophil/lymphocyte ratio in peripheral blood of patients with gastric cancer was related to the decrease of total survival time and disease-free survival time.We found that NLR value was not related to tumor stage and differentiation,but the total survival time and disease-free survival time of patients with high NLR group were shorter than those with low NLR group.?Results?1.The effect of Met+neutrophils on the growth and metastasis of gastric cancer1.1 Compared with met neutrophils,Met~+neutrophils significantly inhibited the activity and proliferation of gastric cancer cells in CCK8 experiment and plate cloning.1.2 SGC7901 gastric cancer cell line co-cultured with Met~+neutrophils had G2phase arrest.Flow cytometry showed that Met~+neutrophils significantly promoted the apoptosis of gastric cancer cells1.3 Transwell test showed that the invasion ability of gastric cancer cells co-cultured with Met~+tans was significantly weakened.24 hours and 48 hours later,the migration and repair ability of gastric cancer cells co-cultured with Met~+tans were weaker.2.The role of HGF/Met signaling pathway in TANs polarization2.1 The expression levels of ICAM-1,Fas,TNF-?,CASP3,i NOS,which were detected by Met~+neutrophils in gastric cancer cell culture supernatant group and HGF group were significantly up-regulated compared with those in the control group,and the expression levels of these cytokines were significantly decreased after adding met antagonist.2.2 Flow cytometry showed that there were more apoptosis in gastric cancer cells co-cultured with Met+neutrophils in the supernatant group and HGF group,and met antagonists could reverse the trend of apoptosis.3.The clinical significance of peripheral blood neutrophils in predicting the prognosis of gastric cancerClinical correlation analysis showed that the increase of the ratio of neutrophils to lymphocytes in peripheral blood of patients with gastric cancer was related to the decrease of tumor stage,total survival period and disease-free survival period.Gastric cancer patients undergoing chemotherapy were divided into high NLR group and low NLR group,and the clinical characteristics of tumor stage,differentiation and survival period were analyzed.We found that NLR value was not related to tumor stage differentiation,but the total and disease-free survival time of patients with gastric cancer in high NLR group was shorter than that in low NLR group.?Conclusion?1.Met~+neutrophils significantly inhibit the growth,invasion and metastasis of gastric cancer.2.Met is a potential surface marker of N1 tumor associated neutrophils.N1neutrophils play a role in killing gastric cancer cells through HGF/MET pathway.3.The ratio of neutrophils/lymphocytes in peripheral blood is related to the progression of gastric cancer,which can predict the survival time of patients with gastric cancer undergoing chemotherapy. |
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