The Function Of Periplaneta Americana Extract In Antitumor Immune Regulation

ObjectiveCancer has become one of the serious diseases which threat to human health.But at the moment, the drugs of strong effection and low toxicity are lack.This study will separate small peptides CII-3 which have anti-tumor activity from periplaneta americana and US crude cockroach fine, used in tumor bearing mice model MFC.by detecting and analyzing the tumor microenvironment and the inherent regulation of immune cells, to assess the role and status of periplaneta americana small peptides CII-3 and US crude refined cockroach in its immune regulation of antitumor.Providing the basis for the development of basic and clinical application of the drug.MethodsTo observe the function of CII-3 and US crude refined cockroach by the improved MTT method in many kind of mice vitro tumor cell proliferation, initially identified mouse tumor cell sensitive to the CII-3 and US crude refined cockroach. Observe the effect of CII-3 and US crude refined cockroach role of complement hemolytic activity by serum total complement activity test.The gastric cancer cells MFC were seeded in 6-week-old BALB/c mice, resting for about 10 days,suggesting that the model was successfully constructed when the right armpit of mice had significant tumor. The mice were divided into six groups randomly :model group,CTX group, CII-3 high / low-dose group, the United States cockroach fine and normal control group, killed after continuous administration of 10 d, and then measured the levels of Ig G、Ig M、Ig A in serum by ELISA after blooding from orbital venous.Taked the peritoneal macrophage and assayed its phagocytosis;Taked the tissue of tumor for measuring the tumor inhibition rate; determined the spleen index,the NKcells by staining CD3-APC/CD49-PE/CD335-FITC with the single cell suspension of spleen, the Dendritic cells by staining with ly71(F4/80)-FITC/CD11c-PE/CD3-APC.determined Tumor-infiltrating T 、 B cells by staining B220-PE/CD3-APC/CD40-FITC with the tumor single cell suspension; To determine the Treg cells by staining CD3-FITC/CD4-APC/Fox P3-PE, to determine the Failure type T cells by staining CD3-FITC/CD8-Per CP/PD1(CD279)-PE/CD4-APC, to determine the TAM, M1 and M2 cells of local tumor microenvironment by staining F4/80-FITC/CD206-PE/CD16/32-PE, to explore CII-3 US cockroach fine antitumor mechanism by flow cytometry analysis.Results1. In vitro studies, it is showed that : in cell toxicity studies, the median inhibitory concentrations(IC50) of both CII-3 and American cockroachare to the fine of gastric cancer cells MFC in mice are low. In the study of Activity of promoting complement, the concentrations that CII-3 and US cockroach fine promote 50%hemolysis(CH50) were 44.8 mg / ml and 47.152 mg / ml, both promote its activity effect for the classical pathway of complement activation, of which the activity of CII-3 is strong.2. In studies of preliminary anti-tumor effect, it is showed that:the mice of each group compared with normal control group were splenomegaly, the mice of model group were enlargement most obviously.CII-3 and American cockroach fine group compared with the control group and the CTX group, spleen index increased more significantly, the difference was statistically significant. Tumor mass in the model group than in the control group the other groups are large, CII-3 low-dose group and the tumor mass US cockroach fine group of relatively small and lighter in color. CII-3 and American cockroach fine group compared with the control group,had significantly reduced tumor weight, the difference was statistically significant,with the inhibitory effect of low-dose group CII-3 is the most significant.3. From the results of MFC tumor-bearing mice macrophages,it is showed :compered to the control group, the phagocytic index and phagocytic percentage of CII-3 and American cockroach fine group were increased, the difference was statistically significant; in-flow analysis, CII-3 regulated TAM and M1 up, M2 down;cockroach fine not regulate TAM clearly.4. The findings of MFC tumor bearing mice on the local microenvironment lymphocyte is that : Compared with the control group, in CII-3 and American cockroach fine group, B cells declined, T cells increased; regulatory T cells and failure T cells were both lower, of which the effect of CII-3 high-dose group down the most significantly.5. The results for MFC tumor-bearing mouse spleen cells of the innate immune showed:CII-3 group compared with the control group, NK cells decreased, but not significantly; US cockroach fine group and model group, NK cells increased.CII-3and American cockroach fine group compared with the control group, DC increased and the CII-3 DC group increased more significant.6. The results of MFC tumor-bearing mice serum immunoglobulin showed that: compared with the control group, Ig M and Ig G levels of normal control group are elevated, Ig A did not change significantly; CII-3 group compared with the control group, Ig A 、 Ig M and Ig G levels were increased, the difference was statistically significant; US cockroach fine group compared with the control group, g M levels increased, but the difference was not statistically significant.ConclusionsCII-3 and American cockroach play a significant role in vitro cytotoxicity and complement a strong pro-active.In the anti-tumor effect of preliminary exploration,CII-3 and American cockroach fine have no inhibitory effect on the spleen, but have significant inhibitory effect on tumor growth.By promoting their phagocytic function,adjusted the ratio of M1 and M2 in TAM, reduced tumor-promoting M1 cells,and did not damage anti-tumor M1.By adjusting the proportion of tumor-infiltrating Tand B lymphocyte, upregulated T cells, downregulated B cells; reduced ailure-type T cells, regulated T cells down.By gulating the spleen and the number of DC’s innate immune cells NK cells, a variety of mechanisms to enhance adaptive immunity humoral immune response functions, CII-3 and American cockroach fine play a role of anti-tumor.