1型糖尿病小鼠同基因骨髓移植治疗后的免疫变化

Immune Changes in Type 1 Diabetes Animal Model After Syngeneic Bone Marrow Transplantation

本研究旨在观察同基因骨髓移植(syn-BMT)治疗1型糖尿病(T1D)小鼠前后外周血T淋巴细胞亚群、NK细胞比例的变化,探讨其在小鼠T1D的发生及同基因骨髓移植在诱导T1D小鼠恢复免疫耐受中的作用。选取C57BL/6J小鼠以链脲佐菌素腹腔注射建立T1D模型,并将成模小鼠分为病鼠移植组及病鼠对照组;另取正常C57BL/6J小鼠6只作为正常对照组。病鼠移植组小鼠于成模后第10天行同基因骨髓移植治疗;正常对照组和病鼠对照组不作处理。每10天监测1次正常对照组、病鼠对照组、病鼠移植组空腹血糖;每3天监测1次病鼠移植组小鼠移植后血象。小鼠成模后第10天即移植治疗当天及移植后30天分别取正常对照组、病鼠对照组及病鼠移植组小鼠外周血标本,用流式细胞术测定外周血T淋巴细胞亚群、NK细胞比例。结果显示,syn-BMT后,小鼠空腹血糖逐渐下降,至移植后25天,与正常对照鼠相比无差异,显著低于对照病鼠;移植后小鼠外周血白细胞和血小板计数分别在接受预处理及骨髓移植后3天和6天达最低值,此后逐渐上升,至移植后27天基本恢复正常。糖尿病小鼠外周血CD4+ T淋巴细胞比例、CD4+/CD8+ T淋巴细胞比值和NK细胞比例均较正常对照鼠明显升高(p<0.01),CD8+ T淋巴细胞比例较正常对照鼠显著下降(p<0.01)。syn-BMT后CD4+ T淋巴细胞比例、CD4+/CD8+ T淋巴细胞比值和NK细胞比例较对照病鼠不同程度的降低,CD8+ T淋巴细胞比例较对照病鼠及正常对照鼠明显升高(p<0.01)。结论:同基因骨髓移植可以逆转糖尿病小鼠的高血糖状态。糖尿病小鼠发病初期,外周血CD4+ T淋巴细胞比例、CD4+/CD8+ T淋巴细胞比值和NK细胞比例增高及CD8+ T淋巴细胞比例降低,这些变化可能与糖尿病的发生有关。同基因骨髓移植可以不同程度的逆转糖尿病小鼠的免疫紊乱。

Syngeneic bone marrow transplantation （syn-BMT）, as a novel therapy for type 1 diabetes （T1D）, has been used more and more widely. This study was aimed to detect the changes of peripheral CD4 ^＋ T lymphocytes, CD8 ^＋ T lymphocytes, CD4 ^＋/CD8 ＋ T lymphocytes and NK cells before and after T1D mice were treated with syn-BMT, and to investigate the effects of these cells in T1D and the effects of syn-BMT-inducing immunotolerence. T1D mouse model was established by multiple low dose streptozotocin injection, the syn-BMT was performed on 10 day after the onset of diabetes. The T1D model mice were divided into group of diabetic mice treated with syn-BMT and group of diabetic control mice （DC）, 6 normal C57BL/6J mice were regarded as normal control group （NC）. On 30 day after syn- BMT, peripheral proportion of CD4 ^＋ T lymphocytes, CD8^＋ T lymphocytes, CD4 ^＋/CD8 ^＋ T lymphocytes and NK cells were detected by flow cytometry. These cells of normal control mice （ NC）, diabetes control mice （DC） and diabetes mice treated by syn-BMT were also detected. Blood glucose level in three groups was assayed during the whole observation period. The results showed that syn-BMT could reduce blood glucose level of T1D mice to near normal （p 〉 0.05）. Hematopoietic reconstitution happened in a month. The proportion of peripheral CD4 ^＋ T lymphocytes, CD4 ^＋/ CD8 ^＋ T lymphocytes, NK cells all increased in new-onset diabetic mice （p 〈 0.01 ）, while the proportion of peripheral CD8 ^＋ T lymphocytes decreased （p 〈0.01 ）. On 30 day after T1D mice were treated with syn-BMT, the proportion of peripheral CD4 ^＋ T lymphocytes was significantly lower than that in DC mice （p 〈0.01 ）, but still higher than NC （p 〈 0.05）. The proportion ofCD8^＋ T lymphocytes was higher than that in DC and NC mice （p 〈0.01）. The ratio of CD4 ^＋/CD8 ^＋ T lymphocytes and proportion of NK cells were both obviously lower than that in DC and NC mice （p 〈 0.01 ）. It is concluded that the syn-BMT can reverse hyperglycemia and immune disorder in diabetic mice. On early period of diabetis onset, the proportions of CD4 ^＋ T lymphocytes and NK cells, the ratio of CIM ^＋/CD8 ^＋ T lymphocytes increase, while proportion of CD8 ^＋ T lymphocytes decreases in peripheral blood which mye be associated with onset of diabetes.