非肥胖性糖尿病（NOD）小鼠糖尿病发病过程中 B10细胞水平的研究

Study on the levels of CD19＋CD5＋CD1dhigh B10 cells in NOD mice

目的：观察非肥胖性糖尿病（ non-obese diabetic， NOD）小鼠自然病程不同阶段CD19＋CD5＋CD1dhigh B细胞及Th1、Th17细胞数量的变化，探讨B10细胞与NOD小鼠自身免疫性糖尿病发生的关系。方法采用流式细胞术分别检测4周（A组，n＝10）、8周（B组，n＝10）及糖尿病（C组，n＝10）NOD小鼠及同龄C57BL／6小鼠（对照组，n＝20）脾脏、肠系膜淋巴结、外周淋巴结、胰腺淋巴结中CD19＋CD5＋CD1dhigh B、CD19＋IL-10＋B、CD4＋IFN-γ＋Th1、CD4＋IL-17＋Th17细胞及CD4＋CD25＋Foxp3＋T细胞的比例；分离NOD小鼠胰腺组织作病理切片以评估胰岛炎的严重程度。结果（1）胰腺组织HE切片：A组无胰岛炎，4周；B组发生胰岛炎，8周；C组发生明显胰岛炎且无完整胰岛，糖尿病。（2）不同病程阶段NOD小鼠B10细胞比较：B及C组B10细胞较A组增高，而C组B10细胞则较B组显著下降，差异有统计学意义（P＜0．01）；A组B10细胞各组织间无明显差异（P＞0．05），B组B10细胞以胰腺淋巴结最高，C组B10细胞以外周淋巴结最高且胰腺淋巴结B10细胞较B组显著下降，差异有统计学意义（P＜0．01）。（3）不同病程阶段NOD小鼠效应性T细胞比较：C组Th1及Th17细胞较A、B组显著增加，差异有统计学意义（P＜0．01）。（4）不同病程阶段CD4＋CD25＋Foxp3＋T细胞比较：C组Treg较A、B组无明显变化，且NOD小鼠Treg较同龄C57BL／6小鼠亦无明显变化（ P＞0．05）。结论随NOD小鼠年龄增长及胰岛炎进展，B10细胞存在数量变化及组织分布的差异；B10细胞数量减少可能参与NOD小鼠自身免疫性糖尿病的发生。

Objective To study the alterations of CD19＋CD5＋CD1dhigh B, Th1 and Th17 cells in non-obese diabetic （ NOD） mice and the correlation between B10 cells and type 1 diabetes in NOD mice. Methods Flow cytometry （ FCM） was used to measure the levels of CD19＋CD5＋CD1dhigh B, CD19＋IL-10＋B, CD4＋IFN-γ＋Th1, CD4＋IL-17＋Th17 and CD4＋CD25＋Foxp3＋T cells in NOD mice （ 4 weeks old NOD mice：group A, n=10;8 weeks old NOD mice：group B, n=10; NOD mice with diabetes： group C, n=10） and age-matched C57BL/6 mice （ control group, n=20 ） .Hematoxylin-eosin staining of pancreatic tissues was performed for histopathological assessment of the development of insulitis in NOD mice.Results （1） Histopathological analysis showed that mice from A, B and C groups respectively showed no insulitis, insulitis and obvious insulitis with no intact islets.（2） The highest levels of B10 cells in NOD mice were ob-served in group B, followed by those in group C and group A （P〈0.01）.No significant differences with the levels of B10 cells were found among different tissues of 4 weeks old NOD mice （P〉0.05）.More B10 cells were detected in pancreatic lymph nodes than in other tissues of 8 weeks old NOD mice, the levels of which were also higher than those in pancreatic lymph nodes of mice form group C （ P〈0.01） .The highest levels of B10 cells were detected in peripheral lymph nodes among all tissues samples collected from NOD mice with diabetes （P〈0.01）.（3） The levels of Th1 and Th17 cells in mice from group C were remarkably in-creased as compared with those in mice from group A and B （P〈0.01）.（4） The percentages of CD4＋CD25＋Foxp3＋T cells in mice from group C showed no differences with those in mice from A and B groups. No significant difference with Treg cells were observed between NOD mice and age-matched C57BL/6 mice （P〉0.05）.Conclusion The percentages and distribution of B10 cells in NOD mice changed with age and the development of insulitis.The decrease of B10 cells might participate in the development of type 1 diabe-tes in NOD mice.