耐受调节性CD8^+NKT细胞体外稳定性的研究

Study on the stability of the anergic CD8^+NKT cells in vitro

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。

AIM： To study the CD8^＋NKT cell stability function.METHODS： Splenic lymphocytes from C57BL/J mice were treated with Staphylococcal enterotoxin B（SEB） and then cultured in vitro.Day 10,20,30 and freezen effector cells were harvested and used.The cells were cultured in medium containing ConA and LPS for 72 hours and measured their response to mitogens.The inhibitory action of the effector cells were examined.The effector cells were co-cultured with normal lymphocytes and above mitogens for 72 hours.The cells proliferation was assessed with MTT method.The effector cells were cultured with heterogenic lymphocytes and were assessed with MTT method.The NKT cell subsets among these effector cells were analyzed by flow cytometry.RESULTS： The response of these effector cells to ConA and LPS was significantly decreased compared with that of normal lymphocytes.The A values of cell proliferation were decreased from 0.67 and 0.61 to 0.30 and 0.31,0.28 and 0.20,0.26 and 0.24,0.22 and 0.23（P〈0.05,n=3）.The inhibitory ability of effector cells aginst the response of normal lymphocytes to ConA and LPS were clearly observed.They inhibited the response of normal lymphocytes to above mitogens.And the A values of cell proliferation were decreased from 0.67 and 0.61 to 0.33 and 0.39,0.30 and 0.43,0.36 and 0.43,0.26 and 0.29（P〈0.05,n=3）.The response of effector cells to heterogenic lymphocytes was significantly decreased compared with normal lymphocytes.The A values of cell proliferation were decreased from 0.70 to 0.42,0.42 and 0.54 on day 20,30 and freezen effector cells respectively（P〈0.05,n=3）.The CD8^＋NKT cell subsets among these effector cells with tolerance function were significantly increased（P〈0.05,n=3）.CONCLUSION： The anergic CD8^＋NKT cells could culture in vitro.And the anergic function of these cells were still existing.