Effects Of CD40 Signal Regulating Regulatory B Cells On The Biological Behavior Of Colon Cancer

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Relationship between peripheral blood B cells and regulatory B cells and colon cancerObjective:To analyze the levels of CD19+B cells and CD19+IL-10+Bregs in peripheral blood and the clinical stage of colon cancer and the predictive effect on the risk of recurrence of colon cancer.Methods:Use flow cytometry to test the numbers of CD 19+B cells and CD 19+IL-10+Bregs in peripheral blood of colon cancer patients and healthy volunteers,and then analyze relationship between the numbers of CD 19+IL-10+Bregs and the clinical data of colon cancer.According to the clinical relapse of 24-month follow-up of colon cancers,the relationship between postoperative recurrence and the number of CD 19+IL-10+B cells in the peripheral blood of patients was calculated by Kaplan-Meier method.The Cox regression model was used for multivariate analysis and screening risk factors for recurrence and metastasis.Results:1.The percentages of CD19+B cells in peripheral blood of patients with colon cancer and healthy people were 7.76%VS 6.92%(P=0.240).2.The percentage of CD 19+IL-10+Bregs in CD 19+B cells was significantly higher than that in healthy subjects4.28%VS 0.98%(P=0.004),and the number of CD19+IL-10+Bregs was positively correlated with the colon cancer stage(P<0.05).3.COX proportional hazards model found that the number of CD 19+IL-10+Bregs was an independent predictor of recurrence in patients with colon cancer(risk coefficient OR was 2.159,95%confidence interval 0.272-17.130).Conclusion:The percentage of regulatory B cells in peripheral blood of colon cancer is significantly higher than that of normal persons,and it is related to the TNM stage of colon cancer.Its level may predict the recurrence of colon cancer.Part ? Relationship between regulatory B cells and CD40L in colon cancer patientsObjective:To investigate the correlation between CD40 and regulatory B cells in colon cancer patientsMethods:Detect the serum levels of sCD40L in colorectal cancer patients using ELISA kit,and analyze the correlation between with sCD40L and the number of CD 19+IL-10+Bregs.CD40L in colon cancer tissues was detected by immunohistochemistry and the expression of CD19+IL-10+Bregs in its paracancerous tissues was tested by Flow cytometry.Then analyze the correlation between with CD40L and the number of CD 19+IL-10+Bregs in tissuesResults:1.The level of sCD40L in colorectal cancer patients was2.318±1.201ng/ml,and that of normal healthy human was 1.622±0.680ng/ml.There was significant difference between the two groups(P=0.027).The level of sCD40L in colorectal cancer patients was significantly correlated with the number of CD 19+IL-10+Bregs in peripheral blood,Spearman coefficient was0.763,P<0.01.2.In patients with CD40L positive expression in colon cancer tissue,the expression of CD 19+IL-10+Bregs was 6.33%in his paracancerous tissues,and it was 2.38%in the negative expression of CD40L colon cancer tissue.The difference was statistically significant(P<0.01)Conclusion:There was a positive correlation between the concentrations of soluble CD40L and the level of regulatory B cells in peripheral blood of colon cancer.The level of regulatory B cells in para cancerous tissue with CD40L expression positive in colon cancer tissue was significantly higher than that of in negative CD40L in colon cancer tissue.The result shows that there is a certain correlation between CD40L and regulatory B cells in colon cancer microenvironmentPart ? Relationship between the function of regulatory B cells and CD40Objective:To investigate the effect of CD40 on Bregs self-function and the effect of Bregs on CD4+T cells through CD40 signal pathwayMethods:B cells and CD19+CD5+CD1d+Bregs were extracted from both mice(wild-type WT and CD40 knock-out genotype CD40KO)by Flow cytometry.B cells and CD 19+CD5+CD1d+Bregs were cultured in vitro with CpG-ODN or CD40L and CpG-ODN.The proportion of CD19+CD5+CD1d+Bregs in B cells and the level of IL-10 in CD 19+CD5+CD1d+Bregs culture supernatants were detected before and after culturing.Two kinds of CD 19+CD5+CD1d+Bregs(WT and CD40KO)were respectively co-cultured with CD4+T cells to test the growth of CD4+T cells,the proportions of CD4+CD25+Tregs and the level of IFN-?.Results:1.The percentage of CD19+CD5+CD1d+Bregs in B cells significantly increased in WT B cell culture system with CD40L and CpG-ODN(P<0.01),while the level of CD 19+CD5+CD1d+Bregs in B cells did not change significantly in CD40KO B cell culture system(P>0.05).2.The proliferation of WT CD 19+CD5+CD1d+Bregs was significantly higher than that of CD40KOCD19+CD5+CD1d+Bregs in the culture system with CD40L and CpG-ODNCD40L,and while the level of IL-10 in the supernatant was significantly increased(P<0.01).Compared with CD40KOCD19+CD5+CD1d+Bregs group,WT CD19+CD5+CD1d+Bregs could inhibit CD4+T cell proliferation,IFN-?.secretion(P<0.01)and induce CD4+CD25+Tregs production(P<0.01).Conclusion:Activation of CD40 signaling can induce the production and proliferation of regulatory B cells,and can induce CD4+CD25+Treg cell proliferation inhibit the proliferation of CD4+T cells and IFN-? secretion in IL-10 dependent mannerPart ? Effects of CD40 Regulating Regulatory B Cells on Colon Cancer Tumor-bearing Mice and Its Related MechanismObjective:To investigate whether the regulatory effect of CD40 on regulatory B cells can affect colon cancer-bearing mice and its mechanism.Methods:C57BL/6 mouse tumor model was established by using colon cancer MC38 cells.One week later,tumor-bearing mice were injected with CD19+CD5+CD1d+Bregs from two different mice(WT and CD40KO)by tail veins.Mice were sacrificed after 2 weeks.Remove the tumor and measure the size to calculate the volume.Collect the spleen,and prepare them to suspension of spleen mononuclear cells by cutting,grounding and filtering.The percentages of CD4+CD25+Tregs in CD4+T cells in different groups were compared by flow cytometry.The blood serum of abdominal aorta was left in mice by centrifugation.The level of IL-10 and IFN-? in the serum of mice was measured by ELISA double antibody sandwich method.Results:1.The tumor volume in group WT CD19+CD5+CD1d+Bregs(129.70±48.17 mm3)was significantly larger than that in CD40KOCD19+CD5+CD1d+Bregs group(61.60±37.27 mm3)and the tumor volume in blank control group(41.30±12.51 mm3)(P<0.05).There was no significant difference in tumor volume between the CD40KOCD19+CD5+CD1d+Bregs group and the blank control group(P>0.05).2.The percentage of CD4+CD25+Tregs/CD4+T cells in spleen of WT CD 19+CD5+CDld+Bregs group was significantly higher than that of CD40KOCD19+CD5+CD1d+Bregs group(5.20±0.15%VS 4.94±0.22%)(P<0.01).There was no significant difference in CD4+CD25+Tregs/CD4+T between CD40KOCD19+CD5+CD1d+Bregs group and blank control group(P>0.05).3.The serum IL-10 concentration of WT CD19+CD5+CD1d+Bregs group was significantly higher than that of CD40KOCD19+CD5+CD1d+Bregs group(53.80±3.82pg/ml)and blank control group(75.6±8.04pg/ml)(P<0.01).The concentration of IFN-? in WT CD 19+CD5+CD1d+Bregs group was significantly lower than that in CD40KOCD19+CD5+CD1d+Bregs group(82.40±6.85pg/ml)and blank control group(85.58±5.45pg/ml)(P<0.01).And there were no significant differences in serum IFN-?and IL-10 level between CD40KOCD19+CD5+CDld+Bregs group and blank control group(P>0.05)Conclusion:Activating the CD40 signal of regulatory B cells promotes the tumor growth of tumor bearing mice with colorectal cancer.It suggests that regulatory B cells may break the body's anti-tumor immunity,which involves the interaction between regulatory B cells and T cells,as well as the secretion of related factors IL-10 and IFN-?.