| Objective: There are multiple types of inhibitory immune cells in tumor microenvironment. Among these cells, CD4~+CD25~+T regulatory cell(Treg)is a sort of multi-cytokine secreting (TGF-β,interleukin-4,interleukin-10,et al),affecting T cell suppressing cell, which can mediate immune tolerance and escape. To investigate the impact of radiotherapy on BALB/c mouse model of breast cancer tumor microenvironment in the CD4~+CD25~+Foxp3~+ regulatory T cells (Treg cells), the results of our research may provide experimental support for future CD4~+CD25~+Treg based antitumor immunotherapy.Methods: 4T1 breast cancer cell culture, the purchase BALB /c mice, the establishment of BALB/c mice bearing 4T1 cells (murine breast cancer) model. Cultured 4T1 mouse breast cancer cells were BALB/c mice 5-6 intercostal chest wall skin (2×105 cells /mouse), fixed mouse limbs in the plate, the irradiation field reduced to slightly beyond the tumor size range, with the accelerating voltage 6mV; tissue dose 6000cGy; measured rate 500cGy/min; wavelength of 100cm in the line of radiation dose local tumor irradiation in mice, Every mice do a single radiotherapy treatment ,at the time of before and after radiotherapy 4, 8, 12 days. Take tumour tissues and surrounding normal tissues,then paraffin embedding, sectioning and dewaxing and hydration, applied immunohistochemical kit, do immunohistochemical stains by SP, detect the surrounding tissue and tumor tissue of the Foxp3 expression. Take tumor tissue, do a 200 mesh copper nets grinding, preparation tumor cell levitation liquid, line flow cytometric dyeing, detection CD4+CD25+Foxp3+ Treg content. CellQuest software analysis, with CD4+CD25+Foxp3+/ CD4+CD25+Treg proportion reflect the content of Treg, To observe the effect of specific radiotherapy dose afect the tumor tissue and the surrounding tissue about the Treg content .Results:1 The tumor volume and lymph nodes and growth rate are obviously decreased after radiotherapyBy measuring tumor with caliper length-diameter (a), (b), short diameter every four days,and then by volume (V) =πab2/6 to calculation tumor size, and calculation tumor growth inhibition rate. After local radiotherapy 4,8,12 days, mesure the tumor size and growth rate. The results showed that along with radiation to the extension of time, the radiotherapy of tumor size and growth speed decreased significantly slowed when compared with control group.2 Radiotherapy of tumor lowered the expression level of Foxp3 within the tumor organizationIn order to detect the expression of Foxp3, tumors and normal tissues were taken to do immunohistochemical staining before and after 4,8,12 days radiotherapy. The results showed that, along with days increasing, the expression level of Foxp3 is higher than normal tissue, but after radiotherapy the level of Foxp3 within the tumor tissue descreased markedly, compared with before. The results showed that, the expression level of Foxp3 in the tumor tissue increased than the normal tissue, radiotherapy can make the expression reduced.3 The expression level of CD4+CD25+Foxp3+Treg reduced after radiotherapyTo test Treg content application of software ofCellQuest and flow cytometric dyeing analysis, calculation of mice after a successful 1 days after radiotherapy and eight days and the proportion of CD4+CD25+Foxp3+/ CD4+CD25+Treg. The results show that the expression level of CD4+CD25+ Foxp3+Treg increased slightly on the first day, whereas the level elevated obviously in the tumors and lymph nodes of tumor-burdened mice at the eight day.Compared with radiation group, CD4+CD25+ Foxp3+Treg level is decreased obviously. The results above showed that, in the early and later days, the expression level of CD4+CD25+ Foxp3+Treg (7.07+0.35) increased significantly in the tumor tissue and the drainage of lymph nodes (P < 0.05), radiotherapy reduced the level of the CD4+CD25+ Foxp3+Treg.Conclusion:1 The tumor volume and lymph nodes and growth rate are obviously decreased after radiotherapy2 Radiotherapy of tumor lowered the expression level of Foxp3 within the tumor organization.3 The expression level of CD4+CD25+Foxp3+ Treg reduced after radiotherapy. |
PDF Full Text Request