异基因骨髓干细胞移植后重建NOD小鼠T淋巴细胞亚群比值的研究

PREVENTION OF DIABETES IN NOD MOUSE BY ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

目的:探讨骨髓干细胞移植对NOD小鼠免疫系统重建和预防糖尿病发生的机制。方法:①21只6周龄NOD雌性鼠接受9.5cGy全身照射后随机分为两组,移植组10只,对照组11只;②每周监测血常规和血糖的变化;③小鼠饲养至发生糖尿病时或至30周龄时处死,摘取脾脏分离淋巴细胞,通过流式细胞术检测移植鼠与非移植鼠脾脏CD3+、CD4+、CD8+、CD4+/CD8+淋巴细胞;采用ELISA法测定血清中IFN-γ水平;吉姆萨染色法计数小鼠骨髓分裂相中Y染色体所占比例,检测嵌合程度并作为细胞植活标记。结果:①移植后NOD受鼠经骨髓细胞的性别染色体检测显示均植入成功;②实验组糖尿病发病率显著低于对照组(P<0.05),且发病时间与对照组比较明显延缓(P<0.05);③实验组和对照组CD3+T细胞、CD4+T细胞比较差异无统计学意义(P>0.05),但实验组CD8+T细胞显著高于对照组(P<0.05)。实验组的CD4+/CD8+比值显著低于对照组(P<0.05),血清IFN-γ数值也显著低于对照组水平(P<0.01)。结论:NOD鼠在通过异基因骨髓干细胞移植后,能降低糖尿病的发生率和延缓糖尿病的发生,可能与移植后免疫重建,下调CD4+/CD8+T细胞的比值,使Th1/Th2极化向Th2偏移等机制相关,异基因骨髓干细胞移植可能成为防治1型糖尿病的新途径。

Objective： To investigate the effects of allogeneic hematopoietic stem cell transplantation （HSCT） on modifying T lymphocytes and preventing overt diabetes（or pancreatitis）. Methods：Twenty-one female NOD mice（6 weeks old） were received 9.5 cGy dose total body irradiation （TBI）, then randomly divided into transplantation group（10 mice） and control group（11 mice）. When the mice became overt diabetes or the mice were over 30 weeks old, they were killed and the spleen lymphocytes were collected. The CD3^＋, CD4^＋, CD8^＋T cells proportion were analyzed by flow cytometry（FCS） and serum levels of cytokines IFN-γ/were determined by enzyme-linked immunosorbent assay （ELISA）. Sex chromosomes were detected after Giemsa＇s stained and pancreatitis was determined by hematoxlin-eosin stained. Results： The incidence rate of diabetes in transplantation group was obviously lower than that in untransplantation group （ P〈0.01） ,and the time to be diabetes was delay. ELISA showed that IFN-γ in transplantation group was obviously lower than that in non-transplantation group （ P〈0.01）. The number of CD8^＋T cells in splenocytes in transplantation group was higher than that in untransplantation group and the number of CD3^＋ , CD4^＋T cells was no significant difference between these two groups （ P〉0.05）, but the ratio of CD4^＋/ CD8^＋ of T cells in transplantation group was significant lower than that in untransplantation group（ P〈0.05）. Y chromosome was found in all female NOD mice in transplantation group, while pancreatitis was more milder in transplantation group. Conclusion： Allogeneie bone marrow stem cell transplantation can change cellular immunity, reduce and delay the incidence of diabetes in female NOD mice. The mechanism may be related to the new balance in disordered immune system and the rebuilding cellular immunity, and restore the balance of Th1/Th2. It is suggested that allogeneic bone marrow stem cell transplantation maybe a new way for type 1 diabetes immuno-therapy.