AOPP抑制CD4+CD25+调节性T细胞扩增的分子机制

It has been widely accepted that the number and suppressive capacity of CD4+CD25+ regulatory T cells (Tregs) is affected in patients with end stage renal disease (ESRD), even after they received renal transplantation. Although the accumulation of oxidized LDL or leptin accelerates the apoptosis or decreases the proliferation of Tregs, studies have established that normal numbers of functional Tregs cannot be obtained only by deletion of oxidized LDL and leptin in these patients. These findings suggest that some other uremic solutes that accumulate during ESRD, even after renal transplantation, may be involved in these processes. Our previous study showed that advanced oxidation protein products (AOPP) could inhibit Tregs expanding. Nevertheless, the exact mechanism by which AOPP performs this function remains to be unknown. To address this question, we will isolate Tregs and CD4+CD25- T cells from human peripheral blood, and utilize AOPP, album, or AOPP combined with AOPP antibody, to examine the expanding of Tregs and to explore the related molecular mechanisms. In addition, animal experiment will be carried out to observe the effect of AOPP on the expanding of Tregs in vivo. The aims of this work is to elucidate the role of AOPP in inhibiting Tregs expanding and to provide a novel method to achieve normal numbers of functional Tregs in patients with ESRD, even after renal transplantation, in order to delay the progression of ESRD and gain renal transplantation tolerance.

既往研究无法圆满解释终末期肾病(ESRD)患者（甚至其在接受肾移植后）体内CD4+CD25+调节性T细胞(Tregs)数量减少和功能受损的机制。我们研究发现，晚期氧化蛋白产物(AOPP)抑制Tregs扩增，但其机制并不明确。本研究分离正常人外周血Tregs和CD4+CD25-T细胞于体外培养，采用3H-TdR掺入法、流式细胞仪检测、免疫印迹、RT-PCR等手段，对比观察Tregs和CD4+CD25-T细胞在AOPP、白蛋白、AOPP抗体等因素单独或联合刺激下的细胞增殖反应、细胞诱导转化、细胞凋亡变化、细胞因子分泌、细胞周期相关蛋白及凋亡相关蛋白表达，分析AOPP抑制Tregs扩增的分子机制。此外，做动物实验进一步观察AOPP在机体内对Tregs扩增的抑制作用。该研究有助于采取措施使ESRD患者体内Tregs 数量及功能保持正常或增加，为延缓ESRD疾病进展和诱导肾移植免疫耐受打下基础。

终末期肾病(ESRD)患者体内CD4+CD25+调节性T细胞(Tregs)数量减少和功能受损。作为尿毒症毒素之一，晚期氧化蛋白产物（AOPP）在其中的作用及其机制不清。本研究分离人外周血Tregs、CD4+CD25-T细胞和诱导树突状细胞（DC）于体外培养，采用3H-TdR掺入法、流式细胞仪检测、免疫印迹等手段，观察AOPP、白蛋白、抗氧化剂等因素单独或联合刺激下的细胞增殖反应、细胞诱导转化、细胞凋亡变化、细胞周期相关蛋白及凋亡相关蛋白表达。此外，给大鼠腹腔注射AOPP制作AOPP增多模型，观察AOPP对大鼠体内Tregs的影响。结果显示，在细胞培养液中加入AOPP后，细胞内NADPH氧化酶关键成分P22phox、P47phox与P91phox的表达水平逐渐上升、活性氧（ROS）产生增加，抑制Tregs细胞内p-ERK、p-AKT、p-STAT5活化和促进P27kip1的表达，进而抑制Tregs增殖；下调p-STAT5的表达，抑制CD4+ CD25-T细胞向Tregs转化；激活JNK和caspase-3，进而诱导Tregs凋亡。AOPP通过氧化应激导致大鼠体内Tregs数量减少。总之，AOPP通过氧化应激抑制Tregs扩增，提示针对AOPP所引起的氧化应激采取措施，有助于使ESRD患者体内Tregs 数量及功能保持正常或增加，为延缓ESRD疾病进展提供潜在的治疗手段。