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10 d 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

2、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0.11%分别提高到55.00%和68.95%。大细胞群中CD8+ 和TCRVB8+NKT 细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。SE

3、B活化的CD8+ 和TCRV8+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TCRV8+ NK1.1+的NKT细胞。这些细胞是大体积淋巴细胞, 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 investigating their morp

4、hology and differentiation pathways. METHODS:  Splenic lymphocytes from C57BL/J mice were treated with either Staphylococcal entertoxin B (SEB) or ConA and then cultured in vitro. On day 5 and day 10,  the lymphocytes were harvested. Normal adherent macrophages and normal lymphocytes were

5、harvested on day 0. Expression of CD69 molecule on the cell surfaces of the lymphocytes and large lymphocytes by SEBactivated was measured using staining of fluorescent antibody and flow cytometry (FACS).  And the percent (%) of NKT subsets was determined  and  analyzed  the 

6、; differentiation pathways of the NKT subsets. Appearance of subsets was observed and compared under a light microscope at  ×400 magnification. RESULTS:  Expression levels of CD69 molecule on the SEBactivated lymphocytes and the giant lymphocytes  were significantly increased to

7、55% and 68.95% respectively as compared to the level (0.11%) detected on the normal lymphocytes (both P<0.01;  n=3). The percent (%) of CD8+NK1.1+  and TcRV8+NK1.1+ NKT subsets in the giant lymphocytes was significantly enhanced to 30.29% and 31.48% (84.0 or 38.9 folds) originally from

8、0.36% and 0.81%,  respectively. Based on the cell distribution shown in the upper part of the FACS picture,  they should be largescale selection.  The SEBactivated lymphocytes in size were larger than not only the ConAactivated cells but also the adherent macrophages with an increase

9、of 5 fold observed under a microscope. They are noadherent cells. There were a few granule seen in cytoplasm .  The value of cytoplasm vs nuclei was  less than 1.0. The SEBactivating CD8+ and TcRV8+NKT subsets were not relative to a NK source. They were produced directly from T cells diffe

10、rentiation. CONCLUSION:  The SEBactivating NKT cells are giant lymphocytes in size,  consisting predominantly of CD8+NK1.1+ NKT cells. They are considered as a subsets of T lymphocytes.    KeywordsNKT cell subsets;  superantigen;  cellular morphology;  cell di

11、fferentiationNKT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

12、、 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细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。RPMI1640培养基购自Gibco公司,  ConA购自Sigma公司, 胎牛血

13、清购自天津市灏洋生物制品责任有限公司, IL2由北京四环生物制药有限公司购入; SEB（自主知识产权, 专利号: 00103991.4）; 抗CD3PerCP、 抗CD4FITC、 抗CD8PE、 抗NK1.1APC、 抗TcRV8PE和抗CD69FITC荧光抗体均购自BD公司。    12  方法    121  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5

14、15;109/L个细胞的悬浮液待用。    122  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 1×109细胞/L, 随后分别加入3.5 mL 400 g/L SEB或者10 mg/L ConA, 混合后置37、  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。    123  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

15、/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2×109/L个细胞。取10 mL置入直径玻璃平皿中, 37、 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 。    124  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5×105细胞/（100 L/孔）, 加入0.1 mL SEB(400 g/L) 在37、 50 mL/L  CO2孵箱中培养。取培养第10天的细胞经抗CD3PerCP、 CD4FITC、 CD8PE和NK1.1APC以及CD3PerCP、 CD4FITC、 NK1.1APC和TcRV8PE荧光抗体四染色后, 和CD69PE染色后用流式细胞仪（FACs Calibue BD,  USA）测定, 并分别记录大细胞群和淋巴细胞群中CD6