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目的研究松果体摘除及补充褪黑素对大鼠胸腺CD4+ CD25+调节性T细胞(Treg细胞)产生、输出及其相关基因叉状头/翅膀状螺旋转录子(Foxp3)表达的影响。方法选用雄性清洁级SD大鼠120只,分为正常组、假手术组、松果体摘除组、松果体摘除+褪黑素低剂量组[腹腔注射7.5mg/(kg·d)]和松果体摘除+褪黑素高剂量组[腹腔注射15mg/(kg·d)],术后4、8周取材。应用流式细胞检测技术、RT-PCR法及免疫组织化学染色法观察并分析松果体摘除及补充外源性褪黑素后胸腺及外周血CD4、CD25双阳性细胞数量及胸腺Foxp3表达的变化。结果松果体摘除术后无论4周或者8周胸腺CD4+ CD25+ Treg细胞数量均无明显变化,补充褪黑素后双阳性细胞数量呈时间、剂量依赖性明显增加;松果体摘除术后4周外周血CD4+ CD25+ Treg细胞数量明显增加,但补充褪黑素后恢复正常,而术后8周各组之间无显著性差异;半定量RT-PCR及免疫组织化学结果显示,松果体摘除后4周大鼠胸腺Foxp3表达水平明显升高,补充高低两种剂量褪黑素后均有所下降并达到正常水平,而松果体摘除后8周各组之间Foxp3的表达无明显差异。结论松果体摘除对胸腺CD4+ CD25+ Treg细胞的产生无明显影响,但导致大鼠胸腺Foxp3的转录和翻译明显增加,胸腺CD4+ CD25+ Treg细胞的输出增加,而补充外源性褪黑素后能逆转上述异常,长时间作用则其影响逐渐消失。
Objective To investigate the effects of pineal extration and melatonin on the production and export of CD4 + CD25 + regulatory T cells (Tregs) and the expression of Foxp3 in rat thymus. Methods 120 male SD rats were divided into normal group, sham operation group, pineal extirpation group, pineal clearance + melatonin low dose group [intraperitoneal injection of 7.5mg / (kg · d)] and Pineal mass + melatonin high-dose group [intraperitoneal injection of 15mg / (kg · d)], 4,8 weeks after surgery. The number of double positive cells of CD4 and CD25 in thymus and peripheral blood and the change of Foxp3 expression in thymus were observed by flow cytometry, RT-PCR and immunohistochemistry after pineal excision and exogenous melatonin supplementation . Results There was no significant change in the number of CD4 + CD25 + Treg cells in the thymus at 4 weeks or 8 weeks after pineal excision. The number of double positive cells after melatonin administration increased in a dose-and time-dependent manner. After 4 weeks of pineal excision The number of CD4 + CD25 + Treg cells in peripheral blood increased significantly, but returned to normal after melatonin supplementation, but there was no significant difference between the groups at 8 weeks postoperatively. Semiquantitative RT-PCR and immunohistochemistry showed that after the removal of pineal gland After 4 weeks, the expression of Foxp3 in the thymus of the rats increased obviously. After the two doses of melatonin were added, the levels of Foxp3 decreased and reached the normal level. However, there was no significant difference in Foxp3 expression between the groups after 8 weeks of pineal excision. Conclusion Pineal exclusion has no significant effect on the production of thymus CD4 + CD25 + Treg cells, but the transcription and translation of Foxp3 in thymus of rats increased significantly. The output of thymus CD4 + CD25 + Treg cells increased, but the exogenous melatonin reversed The above anomaly, a long time effect is gradually disappearing.