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目的:探讨免疫抑制剂环孢素A(CsA)对类风湿关节炎(RA)患者体内参与免疫突触形成的巨噬细胞凋亡的影响。方法:将人单核细胞株THP-1(human acute monocytic leu-kemia cell line)诱导分化的巨噬细胞经葡萄球菌肠毒素(staphylococcal enterotoxin B,SEB)(100μg/L)包被后与活化的Jurkat T细胞(human acute T-cell leukemia cell line)共培养促免疫突触形成,加或不加CsA(1 mg/L),置于无血清RP-MI1640培养液诱导凋亡16 h后,以Annexin V-PI染色,用流式细胞术检测巨噬细胞的凋亡情况。临床收集10例健康对照及10例确诊活动期RA患者外周血,采用免疫磁珠法阴性分选CD4+T细胞并将其与诱导分化的巨噬细胞共培养,亦设加或不加入CsA(1 mg/L)组,研究CsA对免疫突触调控巨噬细胞凋亡作用的影响。结果:在细胞株、健康人及RA患者外周血由单核细胞诱导分化的巨噬细胞和CD4+T细胞相互作用形成免疫突触后巨噬细胞的凋亡率分别为(32.9±2.8)%、(24.7±1.6)%、(14.5%±1.2)%,较相应单独培养的巨噬细胞的凋亡率(61.4±2.4)%、(45.5±2.6)%、(22.9±1.5)%显著降低(P<0.05)。而在加CsA组,细胞株来源的巨噬细胞及RA患者来源的巨噬细胞的凋亡率分别为(48.8±2.0)%、(16.9±1.1)%,较相应参与免疫突触形成的巨噬细胞的凋亡率有所升高(P<0.05),而加入CsA对健康对照巨噬细胞凋亡率的影响差异无统计学意义。结论:RA患者外周血诱导分化的巨噬细胞参与免疫突触的形成后可显著抑制其凋亡率,而CsA能减弱突触对巨噬细胞的这一保护功能,起到促进巨噬细胞凋亡的作用,该结论为减少RA炎性因子的分泌、减弱关节破坏提供了有利的理论依据。
Objective: To investigate the effect of immunosuppressive cyclosporin A (CsA) on the apoptosis of macrophages involved in immunological synapse in patients with rheumatoid arthritis (RA). Methods: The macrophages differentiated from human monocytic leukemia line (THP-1) were infected with staphylococcal enterotoxin B (SEB) (100 μg / L) Jurkat T cells were co-cultured to promote the formation of immunological synapse with or without CsA (1 mg / L). After being induced to apoptosis for 16 h in RP-MI1640 serum-free medium, Annexin V-PI staining was used to detect macrophage apoptosis by flow cytometry. Clinically, peripheral blood from 10 healthy controls and 10 patients with confirmed active RA were collected. CD4 + T cells were negatively sorted by immunomagnetic beads and co-cultured with differentiated macrophages. CsA (with or without CsA) 1 mg / L) group, to study the effect of CsA on immune synaptic regulation of macrophage apoptosis. Results: The apoptosis rate of immunoprecipitated macrophages induced by monocyte-induced differentiation of macrophages and CD4 + T cells in peripheral blood of healthy people and RA patients were (32.9 ± 2.8)% , (24.7 ± 1.6)% and (14.5% ± 1.2)% respectively, which were significantly lower than those of the corresponding cultured macrophages (61.4 ± 2.4%, 45.5 ± 2.6% and (22.9 ± 1.5% (P <0.05). However, the apoptotic rate of macrophages derived from cell lines and RA patients was (48.8 ± 2.0)% and (16.9 ± 1.1)%, respectively, higher than that of macrophages corresponding to immunosynapses The apoptotic rate of macrophages was increased (P <0.05), while the effect of CsA on the apoptosis rate of macrophages was not statistically significant. Conclusion: The macrophages induced by peripheral blood of RA patients can significantly inhibit the apoptosis rate after immunosynaptic formation, while CsA can weaken this protective function of macrophages by synapse, which can promote the apoptosis of macrophages This conclusion provides a good theoretical basis for reducing the secretion of RA inflammatory factors and reducing joint damage.