超抗原SEB活化的NKT细胞亚群形态和分化途径的研究.

1、超抗原SEB活化的NKT细胞亚群形态和分化途径的 研究11-01-28 11:56:00 作者：陈钰,郭业磊，钟编辑：studa20【摘要】 目的：以NKT细胞形态和分化途径为焦点,研究超抗原SEB活化的 NKT细胞亚群的特征。方法：C57BL/J小鼠脾细胞分别经SEB或者ConA诱导, 收集体外扩增10d和5 d的淋巴细胞以及获取正常黏附性巨噬细胞。用荧光 抗体染色,流式细胞仪测定淋巴细胞和大淋巴细胞群表面CD69的表达、NKT细胞亚群百分数和不同NKT细胞亚群的分化途径。倒置显微镜 400倍下比较大 淋巴细胞的形态。结果：SEB活化的淋巴细胞和大淋巴细胞表面CD69分子表达由正常值的0.1

2、1%分别提高到55.00%和 68.95%。大细胞群中CD8+和 TCRVB8+NKT3胞亚群的百分数由原始的 0.36%和 0.81%分别提高到30.29%和 31.48%。这些细胞位于流式细胞图的上部，是大体积细胞群。显微镜下显示 SEB活化的大淋巴细胞体积不仅大于 ConA活化的T淋巴细胞,而且比正常巨噬 细胞大5倍以上，是非黏附性细胞。胞内质粒松散、胞质与核之比1。SEB活化的CD8+和TCR/ 8+NKT细胞亚群均由T细胞直接分化而来。结论：SEB活 化 10 d 的 NKT细胞亚群主要是 CD8+ NK1.1+和 TCR/ 8+ NK1.1 +的 NKT细胞。 这些细胞是大体积淋巴

3、细胞，应属于T细胞的亚群。【关键词】NKT细胞亚群；超抗原；细胞形态；细胞分化Abstract AIM: This study is to explore the biological characteristics of NKT subsets through inv estigati ng their morphology and differe ntiati on pathways. METHODS:Sple nic lymphocytes fromC57BL/J mice were treated with either Staphylococcal en tertox in B (S

4、EB) or ConA and then cultured in vitro. On day 5 and day 10,thelymphocytes were harvested. Normal adhere nt macrophages and no rmal lymphocytes were harvested on day 0. Expressi on of CD69 molecule on the cell surfaces of the lymphocytes and large lymphocytes by SEB activated was measured using sta

5、ining of fluoresce nt an tibody and flow cytometry (FACS). And the perce nt (%) of NKT subsets was determ ined andan alyzedthe differe ntiati on pathways of theNKT subsets. Appeara nee of subsets was observed and compared un der a light microscope atx 400 magn ificati on. RESULTS:Expressi onlevels o

6、f CD69 molecule on the SEBactivated lymphocytes and thegia nt lymphocyteswere sig nifica ntly in creased to 55% and 68.95%respectively as compared to the level (0.11%) detected on the no rmal lymphocytes (both P0.01;n=3). The perce nt (%) of CD8+NK1.1+ andTcRVB 8+NK1.1+ NKT subsets in the giant lymp

7、hocytes was significantly enhanced to 30.29% and 31.48% (84.0 or 38.9 folds) origi nally from 0.36% and 0.81%, respectively. Based on the cell distributi on show n in the upper part of the FACS picture,they should be largescaleselectio n.The SEB activated lymphocytes in size were larger tha nnot onl

8、y the ConA activated cells but also the adhere nt macrophages with an in crease of 5 fold observed un der a microscope. They are noadhere nt cells. There were a few granule see n in cytoplasm .Thevalue of cytoplasm vs nu clei wasless tha n 1.0. The SEBactivating CD8+ and TcRV B 8+NKT subsets were no

9、t relative to a NK source. They were produced directly from T cells differe ntiati on.CONCLUSION: The SEB activati ng NKT cells are gia nt lymphocytes in size,con sisti ng predomi nan tly of CD8+NK1.1+ NKT cells. They arecon sidered as a subsets of T lymphocytes.Keywords NKT cell subsets; superantig

10、en; cellular morphology;cell differe ntiati onNKT细胞（nature killer T cell）既表达T细胞受体TCR又表达NK细胞的表面标志CD161（NK1.1）,最初被认定为第4类的淋巴细胞1。目前众多学者 认为NKT细胞应属于T淋巴细胞亚群,是T细胞在特殊状态或者活化状态下特 殊的表达方式2。由于NKT细胞的活化以识别非多态性的 CD1d分子递呈的 糖脂类抗原为特征，在很长的一段时间，人们都认为能够活化NKT细胞的抗原 仅限于a Galcer。随着研究的进展,人们又发现了 NKT细胞可能还有其他 的抗原决定族（ligands ）能够识别

11、不同来源的脂类或者多肽类抗原而被活化3。到目前为止,关于NKT细胞的分型、分群和生物功能以及细胞的形态特征和器官来源等问题都不十分清楚。本研究中对多肽类抗原超抗原金黄色葡萄球菌肠毒素 B（Staphylococcal enterotoxin B,SEB）活化的NKT纟田胞亚群的形态和分化途径做初步研究。1材料和方法1.1 材料 C57BL/J小鼠（雌性，质量1820 g, 810周龄） 来自解放军总医院实验动物中心二级动物房。RPMI164C培养基购自Gibco公司,ConA购自Sigma公司,胎牛血清购自天津市灏洋生物制品责任有限公司 IL 2由北京四环生物制药有限公司购入；SEB （自主知

12、识产权,专利号： 00103991.4）;抗 CD3 PerCP、抗 CD4 FITC、抗 CD8 PE 抗 NK1.1 APC 抗TcRVB8 PE和抗CD69 FITC荧光抗体均购自BD公司。1 . 2 方法1 . 2. 1 正常小鼠淋巴细胞制备摘取C57BL/J小鼠脾细胞在无菌台前分离细胞,用含100 mL/L胎牛血清的RPMI1640培养液制成细胞悬液,经 红细胞裂解，洗涤2次后，制备成密度为5X109/L个细胞的悬浮液待用。1 . 2. 2 SEB或ConA活化的淋巴细胞的制备在25 mL培养瓶中加入3.5 mL 1 X 109细胞/L,随后分别加入3.5 mL 400卩g/L SE

13、B或者10 mg/L ConA,混合后置37C、 50 mL/L CO2孵箱培养10 d （第5天洗涤换液后细 胞继续培养到第10天）和5 d,收获细胞，作为待测NKT细胞。1 . 2. 3 巨噬细胞的制备摘取C57BL/J小鼠脾细胞在无菌台中分离细胞，用含100 mL/L胎牛血清的RPMI1640培养液制成细胞悬液，经红细胞 裂解液裂解,洗涤2次后，制备成密度为2X109/L个细胞。取10 mL置入直径 玻璃平皿中,37 C、50 mL/L CO2孵箱中孵育2 h。去除上清液,用含100 mL/L胎牛血清的RPMI1640培养液洗涤23次。黏附细胞为巨噬细胞，用于与 大细胞的形态比较。1 . 2. 4 SEB活化的淋巴细胞和大细胞表面 CD69的表达及NKT细胞 亚群和分化途径的解析在96孔板中加入5X 105细胞/ (100卩L/孔)，加入0.1 mL SEB(400卩g/L)在37C、50 mL/LCO2孵箱中培养。取培养第10 天的细胞经抗 CD3 PerCP、CD4 FITC、CD8 PE和 NK1.1 APC以及 CD3 PerCP. CD4 FITC、NK1.1 APC和 TcRVp8 PE荧光抗体四染色后, 和CD69 PE染色后用流式细胞仪(FACs Calibue BD