肿瘤坏死因子-α诱导蛋白-8样分子2对小鼠调节性T细胞免疫调节功能的影响  

Regulatory effect of tumor necrosis factor-oL induced protein 8 like-2 expression on immunosuppressive activity of CD4^＋ CD25^＋ regulatory T cells in mice

作　　者：栾樱译[1] 姚咏明[1] 董宁[1] 于燕[1] 

机构地区：[1]解放军总医院第一附属医院全军烧伤研究所基础部,北京100048

出　　处：《中华创伤杂志》2012年第4期316-321,共6页Chinese Journal of Trauma

基　　金：基金项目：国家自然科学基金资助项目（30971192,81130035,81071545）;国家重点基础研究发展计划资助项目（2012CB518102）

摘　　要：目的观察肿瘤坏死因子α诱导蛋白-8样分子2（tumornecrosis factor-αLin．ducedprotein8like-2，TIPE2）在CIM^＋CD25^＋调节性T细胞（CIM^＋CD25^＋Treg细胞）中的表达，并进一步分析其对CIM^＋CD25^＋Treg细胞免疫抑制活性的影响。方法免疫磁珠法分选正常BALB／c小鼠脾脏CIM^＋CD25^＋Treg细胞。采用RT-PCR和Westernblot技术分别从mRNA和蛋白水平检测CD4^＋CD25^＋Treg细胞细胞内TIPE2表达。进一步应用小RNA干扰技术（siRNA）沉默TIPE2表达，流式细胞术观察TIPE2对T淋巴细胞毒性相关抗原（CTLA）-4及又头翼状螺旋转录因子（Foxp3）表达的影响，ELISA法检测CIM^＋CD25^＋Treg细胞分泌IL-10和转化生长因子（TGF）-β水平，并通过MTT比色试验观察效应T细胞增殖活性的改变。结果RT-PCR分析在CIM^＋CD25^＋Treg细胞中检测到147bp大小的特异性TIPE2目的基因条带。Westernblot检测证实CIM^＋CD25^＋Treg细胞中存在清楚的TIPE2条带，相对分子质量为21000。siRNA处理CIM^＋CD25^＋Treg细胞后，经抗CD3／CD28刺激活化的CIM^＋CD25^＋Treg细胞培养24h时CTLA-4和Foxp3表达明显下降（P〈0．01），同时IL-10和TGF-p生成量显著减少（P〈0．01）；此外，CIM^＋CD25^＋Treg细胞对CIM^＋CD25-T细胞的抑制功能明显减弱（P〈0．05）。结论TIPE2作为一种重要的负向调控分子，在CIM^＋CD25^＋Treg细胞中表达并影响CIM^＋CD25^＋Treg细胞的免疫抑制功能。Objective To observe the expression of tumor necrosis factor-αinduced protein 8 like-2 （TIPE2） in CIM ＋ CD25 ^＋ regulatory T cells （CD4 ^＋ CD25 ^＋ Tregs） and analyze its potential effect on immunosuppressive activity of CD4 ^＋ CD25 ^＋ Tregs. Methods CD4^＋ CD25^＋ Tregs were purified from spleen of the BALB/c mice by using magnetic cell sorting system. The expressions of TIPE2 mRNA and protein in CD4^＋ CD25 ^＋ Tregs were detected by using RT-PCR and Western blot. CD4^＋ CD25^＋ Tregs were further infected with the recombinant lentiviruses that carried small interference RNA（siRNA） to knock down the TIPE2 expression. Based on the expressions of cell surface molecules including cyto- toxic T-lymphocyte-associated antigen （CTLA）-4 and forkhead/winged helix transcription factor p3 （ Foxp3 ） detected by flow cytometry and the levels of cytokines including interleukin （IL） -10 and trans- forming growth factor （TGF） -β6 examined by ELISA in CIM ＋ CD25 ^＋ Tregs, the functional role of TIPE2 in controlling suppressive activity of CIM ^＋ CD25 ^＋ Tregs was analyzed. In the meantime, the proliferation activities of T effector cells were assayed by MTT^＋ test. Results A 147 bp TIPE2 gene band and a clearTIPE2 band with a molecular mass of approximately 21 000 in CD4^＋＋ CD25 ^＋ Tregs were detected by using Western blot. Cell surface molecule as well as cytokine expressions were significantly down-regulated when the CD4 ^＋ CD25^＋ Treg stimulated and activated by anti-CD3/CD28 was cultured for 24 hours after the siRNA silenced CD4 ^＋ CD25 ^＋ Treg cells （ P 〈 0. 01 ）. Meanwhile, the suppression role of CD4 ^＋ CD25 ^＋ Tregs on the proliferation activity of T effector cells was weakened obviously （P 〈 0.05 ）. Conclusions As an important immunoregulatory molecule, TIPE2 not only expresses in the CD4 ^＋ CD25 ^＋ Tregs, but also affects the immunosuppressive function of CD4 ^＋ CD25 ^＋ Tregs.

关 键 词：免疫 细胞 肿瘤坏死因子Α 小鼠 蛋白-8样分子2 

分 类 号：R285.5[医药卫生—中药学]