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目的通过观察西罗莫司或环孢霉素A与CD4+CD25+调节性T细胞((regulatory T cell,Treg)体外共培养对CD4+CD25+Treg增殖情况及对Foxp3和转化生长因子-β(transforming growth factor-β,TGF-β)的表达的影响,探讨西罗莫司促进TGF-β分泌诱导Treg体外分化和增殖的机制。方法无菌条件下取C57BL/6小鼠脾脏,分离单个核细胞,免疫磁珠分选获得CD4+CD25+Treg,设空白对照组、西罗莫司组、环孢霉素A组,共培养96h。上流式细胞仪检测CD4+CD25+Treg。反转录聚合酶链、酶联免疫吸附试验法检测西罗莫司或环孢霉素A处理后的CD4+CD25+Treg的FoxP3+、TGF-βmRNA表达水平和分泌情况;Western blot分析TGF-β信号通路重要的活化分子Smad蛋白的表达情况,观察其对CD4+CD25+FoxP3+Treg增殖的影响;使用TGF-β中和抗体进一步验证TGF-β在西罗莫司促进CD4+CD25+FoxP3+Treg分化增殖过程中的重要作用。结果与对照组相比,西罗莫司处理的CD4+T细胞分泌的TGF-β水平增加近2.5倍,差异有统计学意义(P<0.01);而环孢霉素A处理的CD4+T细胞分泌的TGF-β水平则有所下降,但与对照组比较,差异无统计学意义(P>0.05)。与对照组比较,环孢霉素A组CD4+CD25+Treg占CD4+T细胞比例明显降低,差异有统计学意义(41.25%vs.69.22%,P<0.01);西罗莫司组CD4+CD25+Treg占CD4+T细胞稍有下降,但差异无统计学意义(65.21%%vs.69.22%,P>0.05)。西罗莫司组FoxP3+T细胞的比例明显高于对照组,差异有统计学意义(53.7%vs.40.2%,P<0.05);而环孢霉素A组FoxP3+T细胞比例明显低于对照组,差异有统计学意义(23.6%vs.40.2%,P<0.01)。结论西罗莫司在体外培养促进CD4+CD25+Treg的增殖与生长,而环孢霉素A体外抑制CD4+CD25+Treg的增值与生长。西罗莫司通过诱导TGF-β表达分泌来促进CD4+CD25+FoxP3+Treg的增殖。
Objective To investigate the effects of sirolimus or cyclosporine A on the proliferation of CD4 + CD25 + Treg and the effects of Foxp3 and transforming growth factor-β (Treg) on the proliferation of CD4 + CD25 + regulatory T cells (Treg) To investigate the mechanism of sirolimus in promoting the differentiation and proliferation of Treg induced by TGF-β in vitro.Methods C57BL / 6 mouse spleens were isolated under sterile conditions and single nuclei were isolated CD4 + CD25 + Tregs were obtained by cell sorting and immunomagnetic bead sorting. The control group, sirolimus group and cyclosporin A group were co-cultured for 96 hours. The CD4 + CD25 + Tregs were detected by flow cytometry Polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) were used to detect Foxp3 +, TGF-βmRNA expression levels and secretion of CD4 + CD25 + Tregs treated with sirolimus or cyclosporin A; Western blot analysis of TGF-β signaling pathway was important The effect of TGF-β on the proliferation and differentiation of CD4 + CD25 + FoxP3 + Tregs was observed by using TGF-β neutralizing antibody The important role in the process.Results Compared with the control group, the sirolimus-treated CD4 + T cells (P <0.01). However, the level of TGF-β secreted by cyclosporin A-treated CD4 + T cells was decreased, but compared with the control group (P> 0.05) .Compared with the control group, the proportion of CD4 + CD25 + Treg in CD4 + T cells in the cyclosporin A group was significantly lower than that in the control group, with significant difference (41.25% vs.69.22% P <0.01). The percentage of CD4 + CD25 + Tregs in the sirolimus group was slightly decreased, but the difference was not statistically significant (65.21% vs.69.22%, P> 0.05) + T cells was significantly higher than the control group (53.7% vs.40.2%, P <0.05), while the proportion of FoxP3 + T cells in the cyclosporine A group was significantly lower than that in the control group (23.6% vs.40.2%, P <0.01) .CONCLUSION Sirolimus can promote the proliferation and growth of CD4 + CD25 + Treg in vitro, but the increase of CD4 + CD25 + Treg in vitro Sirolimus promotes the proliferation of CD4 + CD25 + FoxP3 + Treg by inducing the secretion of TGF-β.