| Pancreatic cancer is of high malignancy,and it is predicted that the mortality rate of pancreatic cancer will increase year by year in the next decade.Pancreatic cancer is prone to early metastasis.Most patients with pancreatic cancer have distant metastasis at the time of treatment.Most of them have metastasis to the peritoneum to produce ascites and intestinal obstruction.The production of ascites in pancreatic cancer will increase the body load.Therefore,the mechanism and treatment of pancreatic cancer ascites have become an urgent problem to be solved.Malignant ascites contains a large number of immune cells and immune factors,and these immune cells and immune factors play an important role in the occurrence and development of malignant ascites.IL-9 is a multi-effect immune effector with dual effects of pro-inflammatory or anti-inflammatory,can promote tumor progression or inhibit tumor progression,so that to be a research hot issue in recent years.The previous study of our group has found that the levels of IL-9 in the ascites of patients with liver cancer is higher than that with liver cirrhosis,the higher levels of IL-9 in the ascites,the shorter the patient's survival period.Previous studies demonstrated that IL-9 can promote the migration and invasion of liver cancer cells and pancreatic cancer cells.Anti-IL-9 antibody was used to neutralize IL-9 in ascites of liver cancer mice could prolong the survival period of mice.Though levels of IL-9 are elevated in ascites of liver cancer,the levels of IL-9 in ascites of pancreatic cancer is unknown.Therefore,it is essential to establish a suitable murine model of pancreatic cancer ascites to clarify the relationship between IL-9 and pancreatic cancer ascites,and to further elucidate the pathogenesis,occurrence and prognosis of pancreatic cancer ascites.What is the level of IL-9 in the ascites of pancreatic cancer?Can IL-9 be used as a therapeutic target for immune intervention in ascites of pancreatic cancer?In order to explore the problems above,we will establish a murine model of pancreatic cancer ascites and detect the levels of IL-9 in peripheral blood and ascites of model mice,and intervene murine models as well.Part one:Establishment of murine model of ascites in pancreatic cancerObjective:Pancreatic cancer is of high malignancy,and it is easy to invade and metastasize to the abdominal cavity to produce ascites,which seriously affects the quality of life and survival time of patients.Therefore,it is particularly important to establish a suitable preclinical murine model,which can be used to elucidate the mechanism and provide a appropriate model for diagnosis and therapy of pancreatic cancer ascites.Methods:Ascites was produced by intraperitoneal injection of C57BL/6mice with the Panc02 cell line?G0?.The first generation mice injected with the Panc02 cell line were named g1.The ascites was then withdrawn from g1 and reinjected into the new mice abdominal cavity,named g2.Loop like this until g10.The proportion of ascites,ascites volume and survival time of mice were compared between g1,g5 and g10.Pancreatic cancer cells were isolated from g1,g5 and g10 ascites mice for in vitro culture,named G1,G5 and G10 respectively.C57BL/6 mice?5 per group?were challenged with 5×105,1×106,2×106 and5×106 per mouse of G0,G1,G5 or G10 pancreatic cancer cells intraperitoneally to select the optimal dose of cells to establish pancreatic cancer ascites murine model.The morphological changes between the cancer cells of G0,G1,G5 and G10 were compared by hematoxylin-eosin?HE?staining and transmission electron microscopy?TEM?.The wound-healing assay and Transwell assay were performed to compare the ability of migration and invasion among G0,G1,G5and G10 cancer cells.The levels of IL-9 in the plasma of normal control mice,also plasma and ascites of g1,g5 and g10 mice were determined by enzyme-linked immunosorbent assay?ELISA?.Results:When further generation of ascites mice developed,the proportion of ascites and ascites volume increased gradually.In contrast,the survival time of mice decreased gradually,the proportion of ascites,ascites volume and survival time of mice were stable until g5 mice.The optimal choice to establish a pancreatic cancer ascites murine model is that 1×106 per mouse of G1 cells intraperitoneal injection.Results of cells morphology,wound-healing assay and TranswellTM assay indicate that the ability of migration and invasion of G1,G5and G10 cells increased gradually compared with G0.IL-9 levels of plasma in ascites mice were higher than that of normal control mice,with the further generation developed,the IL-9 level in the ascites of model mice increased gradually,and the IL-9 levels was negatively associated with the survival time of mice.Conclusion:The optimal method to establish a pancreatic cancer ascites murine model is that 1×106 per mouse of G1 pancreatic cancer cells intraperitoneal injection.IL-9 may promote the development of pancreatic cancer ascites.Part two:Effects of antiIL-9 antibody on pancreatic cancer ascites in miceObjective:To determine the level of IL-9,VEGF and MMP-2 in ascites of murine model mice.To explore the effect and mechanism of anti-IL-9 antibody in the treatment of ascites in pancreatic cancer,and to elucidate the role of IL-9in pancreatic cancer ascites.Methods:The murine model of pancreatic cancer ascites was established by using the G1 pancreatic cancer cells in part one.After 24h of modeling,15mice were randomly divided into 10?g group,20?g group and 30?g group,with5 mice in each group.Three groups were given intraperitoneal injection of 10?g,20?g and 30?g anti-IL-9 antibody respectively for every other day for 5injection.At the end of 24h after the last administration,the mice were sacrificed and the IL-9 concentration in the ascites of each group was detected.The best concentration of anti-IL-9 antibody was selected for later intervention.In the same way,45 mice were modeled and randomly divided into blank control group,negative control group and experimental group,with 15 mice in each group.The experimental group were intraperitoneally injected with 20?g anti-IL-9 antibody,the negative control group was given the same type of IgG antibody injection,and the blank control group was given normal saline injection every other day for a total of 5 injections.10 mice of each group were sacrificed randomly at 24h after the last injection,the volume of ascites was measured,ascites supernatants were collected and the levels of IL-9,VEGF and MMP-2 were detected by ELISA.The rest mice were recorded for survival time.Results:The ascites volumes of the blank control group,the negative control group and the experimental group were?2.87±0.20?ml,?2.88±0.25?ml and?2.17±0.35?ml,respectively.Compared with blank control group and negative control group,the ascites volume of the experimental group was significantly reduced,and the difference was statistically significant?P<0.05?.The expression levels of IL-9,VEGF and MMP-2 in the ascites of each group were compared.The results showed that compared with the blank control group and the negative control group,the IL-9 level in the ascites of the experimental group was significantly lower,and the difference was statistically significant?P<0.05?.Compared with the blank control group and the negative control group,the VEGF level in the ascites of the experimental group was significantly decreased,the difference was statistically significant?P<0.05?.However,the levels of MMP-2 in the ascites of the three groups of mice did not change significantly,and the difference was not statistically significant?P>0.05?.Conclusion:Anti-IL-9 antibody can effectively inhibit the generation of the ascites in pancreatic cancer ascites mice and prolong the survival time of mice.The mechanism by which anti-IL-9 antibodies inhibit ascites in pancreatic cancer may be achieved by inhibiting the expression of VEGF in ascites. |
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