Study On The Impact Of CD4+CD25+T Regulatory Cell By Single Low-dose Cyclophosphamide

Backgrand and objectiveRegulatory T cells(CD4+CD25+T cell) account for 5% to 10% of the CD4+T cell population and constitutively express CD25. CD4+CD25+T cell control key aspects of tolerance to self-antigens. Because tumor-associated antigens are derived from self-antigens, CD4+CD25+T cell may be partially responsible for the lack of antitumor immune response. It has been show that increasing tumor burden is associated with a drastic increase in the proportion of CD4+CD25+T cell. In animal models it has also been shown that the removal of CD4+CD25+T cell enhances antitumor immune responses.It is known that, besides its direct cytotoxic effect as an alkylating chemotherapeutic agent, cyclophosphamide also has immuno-modulatory effects, such as depletion of CD4+CD25+ regulatory T cells. In this kind of respect, we have investigated the optimal dose of and the action time of cyclophosphamide for a long time. It has been show that treating mice with a low dose of cyclophosphamide led to a decrease which peaked at a 50% drop in the number of spleen cells at day 4; by 10 days after cyclophosphamide administration, the spleen cell number was back to pretreatment levels.This is the investigation about cyclophosphamide and CD4+CD25+T cell, and we designed this experiment based of it. It is totally about animal experiments.The abstract objective is in this study, The effects of single low-dose cyclophosphamide(CY) on CD4+CD25+T cell and the tumor generation growth in tumor-implanted mice were researched.MethodsThere were two parts in our experiment. Part A was designed for some mices treated with low dose of cyclophosphamide only and the others which was treated with NS. only. They were called the CY group and the control group . 4 days after cyclophosphamide treatment, the protein expression of scurfin was checked by western blot ; in the mean time, the relative amount of cd4+cd25+Tregulatory cell were detected by using flow cytometry. Then we had a comparison between the CY group and the control group. Part B was designed for the mice which was inoculated with tumor cells within 24h after cyclophosphamide and 00 treatment. And we called them CY+tumor group and 00+tumor group. The general status of mice was observed everyday. We also investigate the effects of single low-dose cyclophosphamide on tumor incidence and tumor weight. The tumor growth and weight loss of mice were monitored to evaluate the efficacy and toxicity of different groups. At the 20^th day of post inoculation ,all mice were killed. Tumors weight were checked, ConA stimulated proliferation was tested by MTT assay. Statistical analysis was performed with SPSS10.0 software.Results1. Compared with the number of relative amount of CD4+CD25+T regulatory cell in control group, that in CY group is (1.651±0.447)%, which is decreased significantly . That means the relative amount of CD4+CD25+T regulatory cell decreased significantly after cyclophosphamide injection.2. Western blot analysis showed that protein expression of scurfin in cyclophosphamide treated group decreased significantly compared with the control group.3. The tumor growth curve in CY+tumor group is near flat and that in NS.+tumor group is very steep, the day of tumor happen of CY+tumor group was postponed.4. Mice in CY+tumor group have slight weight loss and in good status. Mice weight in NS.+tumor group increased steadily and decreased at the end because of heavy tumor burden.5. Compared with the NS.+tumor group, lymphocyte proliferation induced by ConA in CY+tumor group was significantly improved.Conclusion1. Single low-dose cyclophosphamide can decrease the relative number of CD4+CD25+T cell in normal mices effectively.2. Single low-dose cyclophosphamide can decrease the expression of scurfin protein in nomal mice effectively.3. Single low-dose cyclophosphamide can supress the growth of RMA lymphocyte tumor.4. Single low-dose cyclophosphamide can make the day of tumor happened delay.