孕鼠接触SEB对其子代鼠Treg细胞的程序效应及机制研究

Microbial infection during pregnant is an important reason to cause the abnormality of neonates by 5.6% every year in China. Our previous studies showed that maternal staphylococcus enterotoxin B (SEB) administration during pregnancy could affect the percentages of central or peripheral T cells in offspring rats. However, little is known whether or not maternal SEB administration during pregnancy has an impact on offspring CD4+CD25+ Treg cells. In this project, a series of studies will be carried out to examine the changes of Treg cells of neonatal and adult offspring rats after pregnant rats are injected intravenously with SEB on day 16 of gestation. Flow cytometry will be used to determine the percentage of CD4+CD25+ Treg cells, and real-time PCR to quantify FoxP3 mRNA as well as Western blot for FoxP3 protein in the thymus and spleen of both neonatal and adult offspring rats. The levels of both IL-10 and TGF-β in the plasma will be determined by ELISA. Furthermore, the suppressive effect of CD4+CD25+ Treg cells on proliferation of CD4 T cells in vitro will be examined in adult offspring rats. Meanwhile, methylation of FoxP3 gene will be analyzed by MeDIP-qPCR in the thymus and spleen of both neonatal and adult offspring rats. These studies will not only have important implications for understanding the effects of staphylococcal infection during pregnancy on CD4+CD25+ Treg cells of offspring, but also shed light on the pathogenesis of some congenital diseases related to immunological impairment.

我国每年新生儿出生缺陷率为5.6%左右，其中很大一部分原因归咎于妊娠期微生物感染。我们以往研究发现妊娠期孕鼠给予葡萄球菌肠毒素B(SEB)可明显影响子代鼠中枢及外周CD4/CD8 T细胞的构成比例，但有关妊娠期孕鼠给予SEB对子代鼠T细胞起平衡调节作用的Treg细胞影响的研究国内外尚无报道。本项目拟系统研究妊娠期孕鼠给予SEB对新生及成年子代鼠Treg细胞的程序效应及其机制，用流式细胞术检测子代鼠胸腺、脾脏和外周血中Treg细胞的数量；用分选法及放免法等分析Treg细胞的功能；用ELISA检测外周血中IL-10 和TGF-β的含量；用MeDIP-qPCR检测胸腺及脾脏中FoxP3基因的甲基化水平；用实时定量PCR和Western blot检测FoxP3的mRNA和蛋白水平。其结果将为探讨SEB与有关免疫性疾病的发生机理关系、提高人口健康和出生质量具有极其重要的价值。

本项目通过流式细胞术、MeDIP-qPCR、实时定量PCR、Western blot、放免法和ELISA等方法系统研究了妊娠期孕鼠给予SEB对新生及成年子代鼠Treg细胞的程序效应及其机制。其研究结果为：⑴妊娠期孕鼠接触SEB可明显增加新生子代鼠胸腺、脾脏及外周血中Treg细胞的绝对数，但对其百分比无影响；Treg细胞的特异转录因子FoxP3的mRNA及蛋白表达水平也升高，进一步支持Treg细胞数量的增加；新生子代鼠脾脏Treg细胞对CD4T细胞的抑制能力增强及外周血中IL-10 和TGF-β表达升高，揭示新生子代鼠Treg细胞的功能也增强。这些结果表明妊娠期孕鼠接触SEB可影响新生子代鼠Treg细胞的发育和功能。⑵检测发现妊娠期接触SEB孕鼠的成年子代鼠胸腺、脾脏及外周血中Treg细胞数量、FoxP3表达水平及Treg细胞功能，与新生鼠时期变化相似，揭示妊娠期孕鼠接触SEB可对Treg细胞形成程序效应，将其从新生期保留至成年期；⑶成年子代鼠再次给予SEB刺激时，成年子代鼠胸腺、脾脏及外周血Treg细胞数量、FoxP3的mRNA及蛋白表达水平、胸腺和脾脏Treg细胞抑制能力、胸腺和外周血IL-10 和TGF-β表达水平明显减少，揭示妊娠期孕鼠接触SEB可明显减弱其成年子代鼠Treg细胞对SEB再次刺激的应答反应；⑷ MeDIP-qPCR检测发现妊娠期孕鼠接触SEB可明显降低新生及成年子代鼠脾脏中FoxP3的甲基化水平。总之，妊娠期孕鼠接触SEB可通过减少FoxP3的甲基化水平来介导子代鼠Treg细胞的程序效应。