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目的:探讨超抗原SEB活化的效应细胞参与免疫耐受的NKT细胞亚群及分化特征。方法:采用C57BL/J小鼠脾细胞经SEB诱导,收集体外扩增10d的淋巴细胞为效应细胞,与刀豆蛋白(ConA)、脂多糖(LPS)和白介素-2(IL-2)共同培养3d,测定效应细胞对刺激剂的应答反应能力。在正常小鼠淋巴细胞与上述刺激剂反应的同时添加效应细胞,3d后测定效应细胞抑制正常淋巴细胞对刺激原的应答反应能力。用MTT染色方法记录细胞增殖的A值。以ConA活化的淋巴细胞做参照,用流式细胞术(FCM)解析了耐受性效应细胞中NKT细胞亚群并显示分化来源与功能的相关性。结果:与正常淋巴细胞对抗原应答反应能力相比,SEB活化的效应细胞对ConA、LPS和IL-2的应答反应能力明显降低,细胞生长的A值从正常组的0.80±0.04、0.60±0.03和0.55±0.07下降到0.60±0.05、0.30±0.05及0.27±0.04(P<0.01,n=3)。效应细胞抑制正常淋巴细胞对上述抗原的应答反应,尤其抑制ConA活化的T淋巴细胞的应答反应;A值分别由正常值降低到0.26±0.002、0.48±0.04及0.34±0.02(P<0.01,n=3)。效应细胞中CD4+NK1.1+、CD8+NK1.1+、TcRVβ8+/NK1.1+NKT细胞亚群显著增加(P<0.05和P<0.01,n=4)。ConA活化的淋巴细胞是T淋巴细胞并包括CD4-CD8-/CD3+NK1.1+NKT细胞。结论:超抗原SEB活化的耐受功能与CD4+NK1.1+、CD8+NK1.1+、TcRVβ8+NK1.1+NKT细胞亚群有关,它们由T细胞分化而来。CD4-CD8-/CD3+NK1.1+NKT细胞没有参与耐受性调节。
Objective: To investigate the subsets and differentiation of NKT cells involved in immune tolerance induced by SEB activated effector cells. Methods: The splenocytes of C57BL / J mice were induced by SEB and the lymphocytes cultured for 10 days were collected as effector cells and co-cultured with ConA, LPS and IL-2 3d, determine the responder responsiveness of effector cells to stimulants. Effector cells were added at the same time that normal mouse lymphocytes reacted with the above stimulators, and after 3 days the effector cells were assayed for their ability to inhibit the response of normal lymphocytes to the stimulus. The A value of cell proliferation was recorded by MTT staining. Using ConA-activated lymphocytes as a reference, NKT cell subsets in tolerogenic effector cells were analyzed by flow cytometry (FCM) and the correlation between source and function of differentiation was revealed. Results: Compared with the ability of normal lymphocytes to respond to antigen, the responsiveness of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased. The A value of cell growth was 0.80 ± 0.04 and 0.60 ± 0.03 And 0.55 ± 0.07 to 0.60 ± 0.05, 0.30 ± 0.05 and 0.27 ± 0.04, respectively (P <0.01, n = 3). The effector cells inhibited the response of normal lymphocytes to the above antigens, especially the response of ConA-activated T lymphocytes. The A values decreased from normal to 0.26 ± 0.002, 0.48 ± 0.04 and 0.34 ± 0.02 (P <0.01, n = 3). The number of CD4 + NK1.1 +, CD8 + NK1.1 +, TcRVβ8 + / NK1.1 + NKT cells in effector cells was significantly increased (P <0.05 and P <0.01, n = 4). ConA activated lymphocytes are T lymphocytes and include CD4-CD8- / CD3 + NK1.1 + NKT cells. CONCLUSIONS: The superantigen SEB activation is associated with CD4 + NK1.1 +, CD8 + NK1.1 +, TcRVβ8 + NK1.1 + NKT cell subsets differentiated by T cells. CD4-CD8- / CD3 + NK1.1 + NKT cells did not participate in tolerance regulation.