The Effect Of Periplaneta Americana Extract CⅡ-3 On Differentiation And Developpment Of B Lymphocyte In Mice Bearing MFC Tumor

[Objective]Tumor,especially malignant tumor,has become a disease that seriously affects human health.And people have been searching for an efficient and low-side effect treatment.Polypeptides,characterized by wide source,low molecular weight,high specificity and low toxicity,have been a hot topic of research for many years.Periplaneta amerricana extract CⅡ-3 is a peptide-based substances extract from Periplaneta amerricana by Yunnan Key Laboratory for Biomedical Research and Development of Insects at Dali University,and the results show that it can inhibit the growth of many kinds of human tumor cells,but the antitumor mechanism has not been entirely clear in the body.So in this study,we research the effect of Periplaneta americana extract on differentiation and developpment of B lymphocyte in mice bearing MFC tumor to understand its effect on immune function in anti-tumor effect,to provid experimental basis and data support for the research and development of the drug by establishing the model of tumor-burdened mice used mice gastric cancer cells（MFC）,and then processing those mice with the different concentrations of Periplaneta americana polypeptide CⅡ-3.[Methods]SPF female BALB/c mice with an average body weight of about 20g and aged from 6 to 8 weeks were selected to establish a model of MFC tumor-bearing mice.MFC cells in logarithmic growth phase were inoculated into the right armpit of BALB/c mice.The tumor-burdened mice were randomly divided into model group,CTX group,the Periplaneta Americana peptide CⅡ-3 low,medium and high dose groups.Moreover,the normal control group was set up.The normal group and the model group mice were given normal saline by lavage（20 mL/㎏,once a day）.The CTX group mice were injected with CTX intraperitoneally（45mg/㎏,10mL/㎏,every other day）.The low,medium and high dose of CⅡ-3 groups mice were given CⅡ-3by lavage（100 mg/㎏,200mg/㎏and 400mg/㎏,20 mL/㎏,once a day）.Each group consisted of 12 mice.Mice were given the drug from the next day after the modeling,and were continuously treated for 10 days.Then mice were sacrificed the next day after the last treatment.Getting the bone marrow cell of mice aseptically and then stained by fluorescence antibody,use the FACS Calibur to detect and analyze the developmental stage of B lymphocytes,subsets of progenitor B cells（B220⁺CD43⁺）,immature B cells（B220⁺IgM⁺IgD^-）and mature B cells(B220⁺IgM^+/-IgD⁺)in the bone marrow of mice.The spleen in mice was obtained aseptically and made into a single cell suspension,take part of the cell suspension and fluorescent antibody staining,FACS Calibur detect and analyze the distribution of Fo B cells(B220⁺CD23⁺CD21CD35^int)and MZ B cell(B220⁺CD23^-CD21CD35^hi),the non-categorical memory B cells（B220⁺IgD⁺CD27⁺）and categorical memory B cells（B220⁺IgD^-CD27⁺）.Another part of single spleen cell suspension was used to extract the spleen lymphocytes,and were added LPS develop four days,after fluorescent antibody staining,FACS Calibur detect and analyze the activation of B lymphocyte（CD69⁺）.Mouse lymph nodes were taken aseptically and made into single-cell suspension.After stained by fluorescence antibody,FACS Calibur was used to detect and analyze the distribution of B cells in mouse lymph nodes.Mouse abdominal cavity cells were obtained aseptically.After fluorescence antibody staining,FACS Calibur was used to detect and analyze the distribution of B lymphocytes and the subsets of B1 cells(B220^intIgD^-),B1a cells(B220^intIgD^-CD5⁺CD11⁺)and B1b cells(B220^intIgD^-CD5^-CD11⁺)in the abdominal cavity.SPSS17.0 software was used for statistical analysis of the experimental data,which were all represented by mean±standard deviation.Variance homogeneity test and one-way anova were used for comparison between groups.When pairwise comparison was made between groups and organizations,LSD test was used for homogeneity of variance,and game-howell test was used for uneven variance.P<0.05 was considered statistically significant.[Results]1.Analysis of bone marrow cells in tumor-bearning mice showed that,compared with the normal group,model group and CTX group,CⅡ-3 low,medium and high dose group were able to multiply the proportion of overall B lymphocytes in bone marrow of mice,the influence of low concentration CⅡ-3 is better than the influence of medium and high concentration of CⅡ-3（P<0.05）.Compared with the normal group,model group and CTX group,CⅡ-3 low,medium and high dose group could increase the proportion of subgroup of B cell inculded Pro-B cells,Immature B cells and Mature B cells,and mature B cells have a lager percentage than Pro B cells and immature B cells,the influence of high dose group is superior to the low and middle dose group（P<0.05）.2.Analysis of spleen cells in tumor-bearning mice showed that,compared with the normal group,model group and CTX group,CⅡ-3 low,medium and high dose group were able to increase the proportion of overall B lymphocytes in spleen of mice（P<0.05）.Compared with normal group and CTX group,the proportion of MZ B cells of model group and CⅡ-3 low,medium and high dose group were increased（P<0.05）.Compared with model group and CTX group,the proportion of Fo B cells of CⅡ-3 low,middle and high dose group were increased significantly（P<0.05）.Compared with normal group and model group,the proportion of non-categorical memory B cells and categorical memory B cells of CⅡ-3 low,medium and high dose group were increased（P<0.05）.Compared with the normal group and model group,the proportion of activation of B lymphocytes in CⅡ-3 low,middle and high dose group were increased（P<0.05）.From the overall analysis,the influence of mouse spleen B lymphocyte subsets distribution in the CⅡ-3 low dose group is better than the influence of medium dose group and high dose group.3.Analysis of lymph node cells in tumor-bearning mice showed that,compared with normal group,model group and CTX group,the proportion of B cells in CⅡ-3low,medium and high dose groups were obviously increased,and the influence of CⅡ-3 low dose is better than the influence of CⅡ-3 medium and high doses（P<0.05）.4.Analysis of abdominal cavity cells in tumor-bearning mice showed that,compared with normal group,model group and CTX group,CⅡ-3 low,medium and high dose group were able to increase the proportion of total B lymphocytes in the abdominal cavity of mice and the effect of low concentration CⅡ-3 is better than the effect of medium and high concentration of CⅡ-3（P<0.05）.Compared with the normal group,model group,the proportion of B1 cells and B1b cells in CⅡ-3 low,medium and high dose group were obviously elevated（P<0.05）,but the proportion of B1a cells in CⅡ-3 low,medium and high dose group were down-regulated（P<0.05）,and the role of low dose group is superior to the role of middle and high dose group.[Conclusions]1.Periplaneta americana extract CⅡ-3 can enhance the proportion of B lymphocyte in mice bone marrow.At the same time,it also can promote the differentiation and development of B cells in bone marrow,promote the generation of Pro B cells,and promote the development of B lymphocytes from immature B cells to mature B cells.2.Periplaneta americana extract CⅡ-3 can influence the distribution of B lymphocyte subsets in spleen of mice,improve the overall proportion of B cells,MZ B cells and Fo B cells,non-categorical memory B cells and categorical memory B cells.Additionally Periplaneta Americana refined CⅡ-3 can effectively promote the B lymphocyte activation in mice spleen.3.Periplaneta americana extract CⅡ-3 can up-regulate the proportion of B lymphocytes in lymph nodes of mice.4.Periplaneta americana extract CⅡ-3 can increase the proportion of B lymphocytes in the abdominal cavity of mice,and up-regulate the B1 cells and B1b cells,down-regulate the B1a cells.