| ObjectiveThis study is aimed to set up the murine model of mucosal immunodepression by in situ inoculating C-26 colon carcinoma and repeatedly intragastric administration of cyclophosphamide. Detect some mucosal immunological index to understand the characteristics of these two models better. Then, we observe the effect of Ginsenoside Rg3 on the models, and explore its mechanism of mucosal immunology. We also compared the T cell subset and Thl/Th2 cytokines in the blood and that in mucosa to understand the correlation between the mucosal immune and systemic immune. Foxp3 was detected in the mucosal T lymphacytes to understand the change of T reg cell between different group.There are three parts in the study:1, Set up the mucosal immune deficiency models by in site inoculating C-26 colon carcinoma with or without repeated intragastric administration of cyclophosphamide, and explore the its mucosal immunological characteristics.1.1 Set up the mucosal immune deficiency models by in site inoculating C-26 colon carcinoma with or without repeated intragastric administration of cyclophosphamide.1.2 Study the mucosal immunological characteristics of the models.1.2.1 Establish a method to isolate lymphocytes from different parts of mucosal immune system.1.2.2 Explore the changes of T cell subsets composition in mucosal immune system between every group.2, Study the effect of Ginsenoside Rg3 on mucosal immunologic function of tumor-bearing mouse with or without chemotherapy of cyclophosphamine.2.1 Study the effect of Ginsenoside Rg3 on the T cell subset of different parts of mucosal immune system in tumor-bearing mouse with or without chemotherapy of cyclophosphamine.2.2 Study the effect of Ginsenoside Rg3 on the Foxp3, IL-2, IL-10(Thl/Th2 cytokine profile) in mucosal T lymphocytes in tumor-bearing mouse with or without chemotherapy of cyclophosphamine.3, Study the changes of IL-2, IL-10 level (Thl/Th2 cytokine profile) in blood in different groups. Materials and methodsFemale BALB/c mice were randomly allocated to sham operation group,tumor-bearing group, cyclophosphamide group, ginsenoside Rg3 group andcyclophosphamid with Rg3 group. 1 X 106 C-26 colon carcinoma cells were injectedinto plasma membrane of ceacum to make the in situ colon carcinoma model, andthe normal saline instead of colon carcinoma cells were injected to the mouse ofsham operation group. After the inoculation the mice of cyclophosphamide groupand cyclophosphamid with Ginsenoside Rg3 group were treated withcyclophosphamide from the 13th day to the 17th day. the mice of Ginsenoside Rg3group and cyclophosphamid with Rg3 group were intragastric administratedGinsenoside Rg3 for 17 days. All the mice were killed at 24 hour after the lasttreatment. The area of Peyer's patch were measured and the morphological changeof intestine of mice were observed. The T cells subsets of different parts of mucosalimmune system including IEL and LPL cells and Peyer's patch were separated andthe mRNA expression of IL-2, IL-10 and Foxp3 were detected by flow cytometerand FQ-PCR. The level of IL-2 and IL-10 in the serum were detected by ELISA.Results1 The establishment of mice model of C-26 colon carcinoma with or withoutrepeated intragastric administration of cyclophosphamide and the exploration of themucosal immunological character of the model.1.1 The results of the weight of tumor and the tumor inhibition ratio of cyclophosphamide suggested that the tumor weight in tumor-bearing group was higher than cyclophosphamide group significantly, the tumor inhibition ratio of cyclophosphamide was 37.65% which confirmed that the establishment of mice model of C-26 colon carcinoma in situ inoculation is successful.1.2 The area of Peyer's patch of sham operation group, tumor-bearing.group, cyclophosphamide group mice were compared and the relativity of the tumor weight and area of Peyer's patch of tumor-bearing group mice was analyzed by statistical methods. We found that the area of Peyer's patch of intestine in tumor-bearing group mice decreased significantly, which indicated the mucosal immunity was injuried, in the other hand, the area of Peyer's patch of intestine in cyclophosphamide group mice decreased further, which indicated cyclophosphamide made further injure on mucosal immuniity. The tumor weight of tumor-bearing mice has negative relativity with the area of Peyer's patch. The area of Peyer's patch of mice decreased , with the increase of the tumor, which confirmed the mice of C-26 colon carcinoma with or without repeated intragastric administration of cyclophosphamide could be both regarded as the mice model of mucosal immunological injuried..1.3 The separation methods of lymphocytes of different parts in intestine mucosal immune system were established. We detected the lymphocytes activity and the separation extent of intestine membrane of mice. And the purified lymphocytes were identified by flow cytometer. All the results suggested the lymphocytes of different parts in intestine mucosal immune system were separated successfully, and the methods of separation are steady.1.4 The ratio of CD4 positive lymphocytes of IEL and LPL were significantly decreased in the tumor-bearing group and cyclophosphamide group mice, and the ratio of CD8 positive lymphocytes of IEL and LPL were increased in different extent.1.5 Compared with the sham operation group mice, the expression of IL-2 mRNA in T lymphocytes of mucosal immune system of tumor-bearing group mice decreasedand the expression of IL-10 mRNA increased, which indicated that the Thl/Th2 balance and cytokine network were disorganized in the body of tumor-bearing group mice, the Th2 cytokine dominated here. The expression of IL-2 mRNA in cyclophosphamide group mice mucosa increased a little, which indicated that cyclophosphamide could not only reduce the tumor weight, decrease the burden of the tumor-bearing of the body, but also mhance the Thl response. But the expression of IL-10 mRNA doesn't change in the cyclophosphamide group.2. The effect of ginsenoside Rg3 on the mucosal immunologic characteristics of tumor-bearing mice with or without cyclophosphamide.2.1 Ginsenoside Rg3 has no effect on the weight of tumor. This means that gmsenoside Rg3 will not promote the growth of tumor. At the same time, it can increase the area of Peyer'patch, which decrease as a result of cyclophosphamide intragastric administration.2.2 Ginsenoside Rg3 can increase the amounts of CD4 positive lymphocytes in IEL and LPL, which were decreased in the tumor-bearing group and cyclophosphamide group. It can also decrease the amounts of CD8 positive lymphocytes in LPL and PP in certain intent. But it has no remarkable effect on y5TCR and CD8 positive lymphocytes in IEL.2.3 Ginsenoside Rg3 can increase the expression level of IL-2 mRNA in small intestinal mucosal immunological site. It has some therapeutic effect on the decreasion of IL-2 in the mucosa of tumor-bearing group and cyclophosphamide group mice. Ginsenoside Rg3 has no effect on expression level of IL-10 mRNA.3. The effect of ginsenoside Rg3 on the systemic immune system3.1 The ginsenoside Rg3 has no effect on the CD4/CD8 T cell subset in peripheral blood3.2 The effect of ginsenoside Rg3 on the blood-serum level of IL-2 and IL-10 in tumor-bearing mice with or without cyclophosphamide.3.2.1 In rumor-bearing mice, the blood-serum level of IL-10 increased to a certainextent. Cyclophosphamide and Ginsenoside Rg3 decreased the blood-serum level ofIL-10.3.2.2 There are no significant difference in IL-2 level of the blood-serum betweendifferent groups.ConclusionMice models of C-26 colon carcinoma with or without repeated intragastric admmistration of cyclophosphamide made a lot of morphological changes at macro and micro level in their intestinal mucosa. Certain changes also exist in some cytokines' level of different group. The two models has good representativeness, and can be used as mucosal immune defect model for the study of pharmacology and therapeutic way.Ginsenoside Rg3 can improve the changes of mucosal pathology, T cell subsets and cytokine level in tumor-bearing mice with or without cyclophosphamide, but has no significant effection on tumor. Ginsenoside Rg3 can increase the CD4+ T subset in IEL and LPL in a certain extent. Ginsenoside Rg3 can increase the Foxp3 mRNA level in LPL, but if using together with cyclophosphamide, this effect disappears. It has modulation effect on mucosal immune. And this may be the mechanism for ginsenoside Rg3 improving the symptom of tumor patients with or without chemotherapy.The effects of tumor, cyclophosphamide and Ginsenoside Rg3 on Thl/Th2 cytokines in systemic immune and mucosal immune are alike, but others.is different. The result shows that, mucosal immune is an important parts of whole immune system, on the other hand, mucosal immune also has a certain independency.
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