晚期氧化蛋白产物抑制CD4＋CD25-T细胞向CD4＋CD25＋调节性T细胞转化

Advanced oxidation protein products inhibit the converting of CD4 ＋ CD25- T cells into CD4＋CD25＋ regulatory T cells

目的探讨晚期氧化蛋白产物（AOPP）对CD4＋CD25-T细胞向CD4＋CD25-T调节性T细胞（Tregs）转化的影响及机制。方法将CD4＋CD25-T细胞按1×10^6／孔培养于平底96孔板，依据是否加入AOPP分为3组：对照组（加入人血清白蛋白200μg／m1）；AOPP组（加入AOPP200μg／m1）；抗氧化组（DPI100μmol／L预处理1h后再加入AOPP200μg／m1）。流式细胞仪检测各组细胞表型变化和DHE荧光强度，3H-TdR掺入法检测Tregs对效应T细胞（Teff）的免疫抑制功能，West-ernblot法检测细胞内磷酸化信号转导与转录激活因子5（p-STAT5）的变化。结果AOPP组中CD4＋CD25＋T细胞占（28．6±4．5）％、胞内因子Foxp3在CD4＋CD25＋T细胞中的表达率为（10．7±1．7）％，显著低于对照组细胞的相应值（73．8±10．7）％、（64．3±9．4）％（P=0．003，0．001）；抗氧化组细胞表型分别为（41．3±4.6．4）％、（22．3±3．0）％，转化较AOPP组多（P=0．049，0．004），低于对照组（P=0．011，0．002）。在Tregs：Teff为1：1的情况下，AOPP组Teff的每分钟脉冲数值（CPM）为17521．0±1703．8，显著高于对照组的9932．3±1142．9（P=0．003）；抗氧化组的Teff的CPM为15709．7±1272．9，高于对照组（P=0．004），但与AOPP组比较差异无统计学意义（P=0．214）。AOPP组DHE荧光强度为（163．0±11．5）％，较对照组（51．1±4．0）％高（P=0．000）；而抗氧化组DHE荧光强度（113．4±7．8）％较AOPP组低（P=0．003），高于对照组（P=0．000）。AOPP组p-STAT5表达较对照组低（P=0．006）；而抗氧化组p-STAT5表达较AOPP组显著性上调（P=0．008），但仍低于对照组（P=0．043）。结论AOPP抑制CD4＋CD25-T细胞向Tregs转化，其机制可能是通过氧化应激下调p-STAT5的表达。

Objective To investigate the effect and mechanism of advanced oxidation protein products （AOPP） on the converting of CCD4± CD25- T cells into CD4± CD25- regulatory T cells （ Tregs）. Methods CD4± CD25- T cells were cultured in 96 - well plates （ 1 x 106 cells/well）, and were divided according to the presence of AOPP into 3 different groups： Control group （200 μg/ml serum albumin） , AOPP group （200 μg/ml AOPP）, Antioxidant group （pretreated with 100 μg/ml diphenyleneiodonium （DPI） 1 h before AOPP was added）. Flow cytometry was used to detect the pbenotype of CD4± CD25- T cells and the DHE fluorescence intensity. The induced Tregs were collected and cultured with CD4± CD25-T ceils as the effective cells （Teff） , the incorporation of [3HI thymidine was used to evaluate the suppressive function of iTregs on the Teff. Western blotting was used to observe the expression of phospho- rylated signal transducer and activators of transcription 5 （p - STATS ）. Results In AOPP group, （ 28.6 _± 4. 5 ） % of CIM cells expressed CD25 and （ 10. 7 ± 1.7 ） % of CD4 ~ CD25 ± T cells expressed Foxp3, which were significantly lower than those of Control group [ （73.8 ± 10. 7 ） %, （ 64. 3 ± 9.4 ） % ]（P =0. 003, 0. 001 ）. In Antioxidant group, these values were （41.3 _± 6.4）% and （22. 3 ± 3.0）% , which were significantly more than those of AOPP group （P = 0. 049, 0. 004） , but lower than those of Con- trol group （ P = 0. 011,0. 002 ）. When Tregs and Teff were incubated at a 1 1 ratio, the Counts per minute （CPM） of Teff in AOPP group was 17 521.0 ± 1 703.8, which was significantly more than that of Control group 9 932. 3 ± 1 142. 9 （P =0. 003 ）. The CPM of Teff in Antioxidant group was 15 709. 7 ± 1 272. 9, which was more than that of Control group （ P = 0. 004 ） and no significant difference as compared with AOPP group （P =0. 214）. The DHE fluorescence intensity of AOPP group was （ 163.0 ± 11.5） % , which was higher than that （51.1 ±4. 0） % of Control group （ P =0. 000） ; while the DHE fluorescence intensity （113.4 ± 7.8% ） of the Antioxidant group was lower than that of the AOPP group （P = 0. 003 ） and was higher than that of the Control group （P =0. 000）. Compared with Control group, addition of AOPP signifi- cantly downregulated the expression of p - STAT5 in CD4± CD25- T cells （ P = 0. 006）. After antioxidants was added, the expression of p - STAT5 was significantly upregulated compared with AOPP group （P = 0. 008） and lower than that of Control group （ P = 0. 043 ）. Conclusion AOPP significantly inhibit the con- verting of CD4± CD25- T cells into Tregs through downregulating p - STAT5 via oxidative stress in vitr0.