| [Objectives] To culture and expand antigen-specific CD4+CD25+regulatory T cells, and then observe the immune suppression of CD4+CD25+regulatory T cells. Provide new ideas for immune tolerance in organ transplantation induced by antigen-specific CD4+CD25+regulatory T cells.[Methods] Part1:Isolation of CD4+CD25+Treg:CD4+CD25+Treg were isolated from BALB/c mice spleen in two steps by magnetic bead cell sorting (MACS). The first step was negative selection of CD4+T cells, and the second step was positive selection of CD4+CD25+Treg. The purity and Foxp3expression of isolated CD4+CD25+Treg were detected by flow cytometry.Part2:Culture and expansion of antigen-specific CD4+CD25+Treg:①Isolated C57mice spleen cells, and inactivated by mitomycin C. Then co-cultured with isolated CD4+CD25+Treg (10:1) in RPMI1640medium for14days.②Detected the antigen-specificity of cultured CD4+CD25+Treg by T cell expansion inhibition assay. The experiment was divided into four groups:Group1(experimental group):C57mice CD4+CD25T cells+cultured BALB/c mice CD4+CD25+Treg+C57mice spleen cells; Group2(control group):C57mice CD4+CD25T cells+uncultured BALb/c mice CD4+CD25+Treg+C57mice spleen cells; Group3(blank control group):C57mice CD4+CD25T cells+C57mice spleen cells; Group4(third-party control group):Kunming mouse CD4+CD25T cells+cultured BALB/c mice CD4+CD25+Treg+C57mice spleen cells. Detected the antigen-specificity of cultured CD4+CD25+Treg by comparing the expansion of CD4+CD25T cells in the4groups.③T cell expansion inhibition assay of different CD4+CD25+Treg concentration:co-culture cultured BALB/c mice CD4+CD25T cells and C57mice CD4+CD25T cells with0.5:1,1:1,2:1,4:1,8:1respectively. Observe the expansion of CD4+CD25T cells in every well.④The results were analyzed with SPSS14.0statistic analysis software. Use independent sample t-test to compare the results that are normal distribution between two groups, while use rank sum test to compare the results that are not normal distribution, and significance level a=0.05.[Results] Partl:MACS can isolate CD4+CD25+Treg successfully, with the purity of up to90%. So the method is stable and efficient.Part2:l.The cultivation and expansion of antigen-specific CD4+CD25+Treg:After co-cultured with C57mice spleen cells for14days, the BALB/c mice CD4+CD25+Treg were expanded for10-fold.2. T cell expansion inhibition assay:After co-cultured for72hours, all samples were analyzed in flow cytometry.①The proportion of primary C57mice CD4+CD25T cells in groupl was significantly higher than that in group2(86.0%±3.9%VS.40.6%±1.9%, P<0.05). So the cultured BALB/c mice CD4+CD25+Treg was stronger than fresh BALB/c mice CD4+CD25+Treg in the inhibition of C57mice CD4+CD25T cells expansion.②The proportion of primary C57mice CD4+CD25T cells in groupl and group2were both higher than that in group3(86.0%±3.9%VS6.8%±3.4%and40.6%±1.9%VS.6.8%±3.4%, respectively. P<0.05). So, both the cultured and fresh BALB/c mice CD4+CD25+Treg could inhibit the expansion of C57mice CD4+CD25T cells.③The proportion of primary C57mice CD4+CD25T cells in groupl was higher than the proportion of primary Kunming mice CD4+CD25T cells in group4(86.0%±3.9%VS.33.5%±3.0%, P<0.05). So, the inbibitional effect of cultured BALB/c mice CD4+CD25+Treg in C57mice CD4+CD25T cells expansion was stronger than than in Kunming mice CD4+CD25T cells expansion. According all the results above, we concluded that we cultivated antigen-specific CD4+CD25+Treg using spleen cells successfully.3. T cell expansion inhibition assay of different CD4+CD25+Treg concentration: After cultured for48hours, the proportion of primary C57mice CD4+CD25T cells was11.1%,14.1%,23.1%,40.6%,49.6%, respectively. Thus, with the increase of CD4+CD25+Treg proportion, the inhibition effect is subsequently enhanced.[Conclusions]1.After negative sorting and positive sorting, MACS can isolate high purity of CD4+CD25+regulatory T cells, with the purity of up to90%and more than95%of Foxp3expression.2.This assay cultivate antigen-specific CD4+CD25+Treg using spleen cells successfully. Compared with other ways, this method is more simple and efficient.3.The assay verify the inhibition of CD4+CD25+Treg cells to effector T cells, and this inhibition exists between the allografts. However, for Specific antigen, its inhibition effect is weaker than antigen-specific CD4+CD25+Treg.4. The inhibition effect of CD4+CD25+Treg is affected by the ratio of CD4+CD25+Treg and effector T cells. With the increase of CD4+CD25+Treg proportion, the inhibition effect is subsequently enhanced... |
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