【作者】 邱志勇；

【导师】 韩忠朝；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 目的探讨脐带来源的间充质干细胞(UC-MSC)免疫治疗特发性血小板减少性紫癜(ITP)的临床应用潜能。方法:体外建立UC-MSC与ITP(8例)及遗传性球形细胞增多症(HS)脾切除患者(3例)的脾脏单个核细胞共培养体系。数天后用酶联免疫吸附试验(ELISA)法检测共培养上清液中IgG型抗血小板抗体(PAIgG)含量的变化。同时,用Brdu掺入法研究UC-MSC对其中部分ITP患者血小板反应性T辅助淋巴细胞增殖的调节作用。另分离12例ITP及8例健康供者外周血单个核细胞(PBMCs),与UC-MSC按1 UC-MSC:10 PBMCs比例体外共培养3—4天后,收获悬浮细胞,再经佛波脂(PMA)、离子霉素及布雷菲德菌素A(BFA)联合刺激4小时后,流式细胞仪检测CD4~+T细胞极化状态(Th1/Th2比例)。结果:UC-MSC在体外可增强全部ITP患者脾细胞PAIgG的自发性释放。且在低剂量时(1 UC-MSC:100脾细胞)能抑制血小板抗原诱导的PAIgG释放,而在高剂量(1:10)下转为刺激作用。UC-MSC亦能轻微刺激HS患者脾细胞自发性释放PAIgG,促进1例HS病例血小板抗原诱导的PAIgG升高。另外,UC-MSC还能抑制ITP患者血小板反应性T辅助细胞的增殖,并呈现一定的量效关系。而UC-MSC未能明显影响ITP患者及健康人外周血Th细胞极化状态。结论:UC-MSC可体外影响ITP患者脾细胞释放PAIgG,具体调控机制及临床应用价值尚有待深入研究。更多还原

【Abstract】 Object To investigate the potential immunotherapeutical effects of umbilical cord-derived mesenchymal stem cells(UC-MSC) on patients with idiopathic thrombocytopenic purpura(ITP),an autoimmune disease characterized by autoantibody-mediated destruction of the platelets by macrophages in the reticuloendothelial system.Methods Different doses of UC-MSC were cocultured in vitro with splenocytes isolated from eight cases of ITP patients and three cases of hereditary spherocytosis(HS) patients who experienced splenectomy. After a short term culture,the level of IgG antiplatelet antibody(PAIgG) in supernatants was determined by a competitive micro-enzyme-linked immunosorbent assay(ELISA) method.The proliferation of platelet-reactive CD4~+ T lymphocytes was also measured with Brdu incorporation ELISA method after coculture with UC-MSCs for 7 days. In the T subsets experiment,peripheral blood mononuclear cells(PBMCs) were isolated from another 12 chronic ITP patients and 8 healthy donors. After cocultured with UC-MSCs(at the ratio of 1UC-MSC:10 PBMCs) for 3-4 days in vitro,PBMCs were harvested and activated by the combination of PMA,ionomycin and BFA.The ratio of Th1/Th2 was studied by analysis of intracellular cytokines with flow cytometry. Results UC-MSC could significantly promote the spontaneous secretion of PAIgG by splenocytes from all ITP patients.In the platelet-inducing condition,UC-MSC inhibited the production of PAIgG in splenocytes of ITP patients at a low ratio of 1 UC-MSC to 100 splenocytes,while promoted at 1:10.In contrast,UC-MSC slightly stimulated spontaneous secretion of PAIgG from splenocytes of HS patients.Platelet-induced PAIgG production was enhanced in only one HS sample.In addition,UC-MSC exerted a definitely suppressive effect on the proliferation of platelet-reactive T helper cells in a dose-dependent manner.However,UC-MSCs failed to obviously change the polarization of T cell subsets isolated from PBMCs of ITP patients and healthy donors.Conclusion UC-MSC can regulate IgG antiplatelet antibodies secretion by splenocytes of ITP patients in vitro,and further study about exact modulation mechanism and prospect for clinical application is expected.更多还原