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目的:探讨超抗原SEB活化并扩增的CD8+NKT细胞耐受调节功能的稳定性。方法:超抗原SEB活化并体外扩增的10 d、20 d、30 d和冻存效应细胞被用于本研究。以正常C57BL/J鼠脾细胞为对照,将各个效应细胞与刺激剂刀豆蛋白(ConA)或脂多糖(LPS)共同培养72 h,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各效应细胞,72 h后测定效应细胞抑制正常淋巴细胞对刺激剂的应答反应能力。体外扩增和冻存的效应细胞与异源鼠脾细胞做混合淋巴细胞培养,MTT法测定细胞的增殖情况。效应细胞用荧光抗体染色,用流式细胞术(FCM)解析NKT细胞亚群。结果:与正常淋巴细胞对刺激剂的应答反应能力相比,体外扩增10 d、20 d、30 d和冻存效应细胞对ConA或LPS的应答反应能力明显降低,细胞增殖的A值分别由正常值0.67和0.61分别下降至0.30和0.31,0.28和0.20,0.26和0.24,以及0.22和0.23(P<0.05,n=3)。效应细胞抑制正常淋巴细胞对上述刺激剂ConA或LPS的应答反应,分别由正常值0.67和0.61下降至0.33和0.39,0.30和0.43,0.36和0.43,以及0.26和0.29(P<0.05,n=3)。效应细胞与异源鼠脾细胞反应与对照组相比明显的降低,分别由正常值0.70下降至20 d的0.42,30 d的0.42以及冻存效应细胞的0.54(P<0.05,n=3)。在这群效应细胞中,主要是CD8+NKT细胞,由原始的0.36%增加到41.59%(P<0.05,n=3)。结论:耐受调节性CD8+NKT细胞可以在体外进行传代培养,并且这些传代培养细胞的耐受调节功能依然存在。
Objective: To investigate the stability of the regulatory function of CD8 + NKT cells activated and expanded by SEB. Methods: The superantigen SEB activation and in vitro expansion of 10 d, 20 d, 30 d and cryopreservation effector cells were used in this study. The normal effector cells were stimulated with ConA or LPS for 72 h with normal C57BL / J mouse spleen cells as control, and the response ability of effector cells to stimulants was determined. After the normal mouse lymphocytes reacted with the above-mentioned stimulants, various effector cells were added, and after 72 hours, the responsive cells that the effector cells inhibited the response of normal lymphocytes to stimulants were determined. Expansion and cryopreservation of effector cells in vitro and mixed spleen cells were made mixed lymphocyte culture, MTT assay of cell proliferation. Effector cells were stained with fluorescent antibody and NKT cell subsets were resolved by flow cytometry (FCM). RESULTS: Compared with the ability of normal lymphocytes to respond to stimuli, the ability of response to ConA or LPS was significantly reduced at 10 d, 20 d and 30 d in vitro and the A value of cell proliferation was The normal values of 0.67 and 0.61 decreased to 0.30 and 0.31, 0.28 and 0.20, 0.26 and 0.24, respectively, and 0.22 and 0.23 (P <0.05, n = 3), respectively. Effector cells inhibited the response of normal lymphocytes to the above stimulators ConA or LPS from normal values of 0.67 and 0.61 to 0.33 and 0.39, 0.30 and 0.43, 0.36 and 0.43, respectively, and 0.26 and 0.29 (P <0.05, n = 3 ). The response of effector cells and allogeneic splenocytes was significantly decreased from 0.70 to 0.42 at 20 days, 0.42 at 30 days and 0.54 (P <0.05, n = 3) . In this group of effector cells, mainly CD8 + NKT cells, from the original 0.36% to 41.59% (P <0.05, n = 3). Conclusion: Tolerance to regulatory CD8 + NKT cells can be subcultured in vitro, and the tolerance regulatory function of these subcultured cells remains.