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背景:趋化信号的免疫学功能已倍受关注,但趋化因子家族的重要成员RANTES对单个核细胞的免疫活化作用及相关作用途径尚未阐明。目的:观察RANTES趋化因子刺激对人外周血单个核细胞的免疫活化作用及分析其作用途径。设计:观察对比实验。单位:扬州大学临床医学院泌尿外科。材料:实验于2004-12/2005-08在美国路易维尔大学免疫学系实验室完成。主要试剂与仪器包括RPMI1640完全培养液,人重组RANTES,抗CD3单克隆抗体,吡咯啉烷二甲基硫脲,CTLA4Ig,LS500型液闪仪及FACSEpicsXL流式细胞仪。方法:分离人外周血单个核细胞,应用不同终浓度的人重组RANTES和/或抗CD3单克隆抗体进行体外刺激,并对增殖反应显著者给予吡咯啉烷二甲基硫脲或CTLA4Ig进行干预。采用3H-胸腺嘧啶核苷掺入法检测单个核细胞增殖程度,应用流式细胞术检测淋巴细胞表型变化。主要观察指标:3H-胸腺嘧啶核苷掺入率、CD4/CD8比值,CD25,CCR5及CD28表达水平。结果:①人重组RANTES终浓度为100,5000μg/L时,诱导单个核细胞增殖反应出现两次峰值。②100μg/L人重组RANTES刺激产生的增殖反应明显高于50μg/L抗CD3单克隆抗体的刺激(P<0.05),但两者无拮抗或协同作用。③吡咯啉烷二甲基硫脲和CTLA4Ig对100μg/L人重组RANTES诱导的增殖反应均能产生抑制作用,并呈现剂量依赖性。④与刺激前相比,人重组RANTES刺激后淋巴细胞CD25表达水平增加(P<0.05),CCR5表达水平降低(P<0.05),CD28表达水平及CD4/CD8比值无明显改变(P>0.05)。结论:RANTES趋化信号具有不依赖于CD3信号、独特的诱导单个核细胞免疫活化的功能,此效应与白细胞介素2信号通路、CD28共刺激通路和核因子κB的活化密切相关。
BACKGROUND: The immunological functions of chemotactic signals have drawn much attention. However, the immune activation of RANTES to mononuclear cells and its related pathways have not yet been elucidated. Objective: To observe the immune activation of human peripheral blood mononuclear cells stimulated by RANTES chemokine and to analyze its mechanism of action. Design: observe the contrast experiment. Unit: Department of Urology, School of Clinical Medicine, Yangzhou University. Materials: The experiment was performed at the Laboratory of Immunology, University of Louisville, USA from December 2004 to December 2005. The main reagents and instruments include RPMI1640 complete medium, human recombinant RANTES, anti-CD3 monoclonal antibody, pyrrolidine dimethylthiourea, CTLA4Ig, LS500 liquid scintillation and FACSEpicsXL flow cytometry. Methods: Peripheral blood mononuclear cells were isolated and stimulated in vitro by human recombinant RANTES and / or anti-CD3 monoclonal antibody at different final concentrations. Interfering with pyrolidine-dimethylthiourea or CTLA4Ig was given to those patients whose proliferative response was significant. The degree of proliferation of mononuclear cells was detected by 3H-thymidine incorporation, and the phenotype of lymphocytes was detected by flow cytometry. MAIN OUTCOME MEASURES: 3H-thymidine incorporation, CD4 / CD8 ratio, CD25, CCR5 and CD28 expression levels. Results: ① When the final concentration of human recombinant RANTES was 100 and 5000 μg / L, there were two peaks in the proliferation of mononuclear cells. ② The proliferative response induced by 100μg / L human recombinant RANTES was significantly higher than that stimulated by 50μg / L anti-CD3 monoclonal antibody (P <0.05), but there was no antagonism or synergism between the two. ③ Pyrrolidine dimethylthiourea and CTLA4Ig can inhibit the proliferation induced by 100μg / L human recombinant RANTES in a dose-dependent manner. (4) The expression of CD25 in lymphocytes increased (P <0.05), the expression of CCR5 decreased (P <0.05), the expression of CD28 and the ratio of CD4 / CD8 had no significant change . CONCLUSION: RANTES chemotactic signal has the unique function of inducing immune activation of mononuclear cells independently of CD3 signaling. This effect is closely related to the activation of interleukin-2 signaling pathway, CD28 costimulation pathway and nuclear factor-kappa B.