【摘要】 目的:通过对NOD/Ltj小鼠在未发病、发病初期与发病末期不同组织器官中CD4~+T、CD8~+T细胞,Th1、Th2、Th17亚群,iNKT细胞频率及亚群,细胞因子、相关转录因子进行观察分析,进一步了解NOD/Ltj小鼠Ⅰ型糖尿病不同发病阶段细胞免疫功能状态。方法:选用雌性NOD/Ltj小鼠为实验对象。血糖仪检测小鼠空腹血糖值,根据尿糖阳性且连续2次≥11. 1 mmol/L作为T1D发病标准将动物分为未发病组、发病初期组、发病末期组。流式细胞技术(FCM)检测各组小鼠外周血、胸腺、脾脏、肝脏中CD4~+T、CD8~+T细胞,Th1、Th2、Th17亚群,iNKT细胞频率及亚群比例以及腹股沟淋巴结CD4~+T、CD8~+T细胞; CBA检测IFN-γ、TNF-α、IL-2、IL-6、IL-17A、IL-4、IL-10; WB检测PLZF、T-bet、GATA-3、ROR-γt。结果:①与未发病组比较,发病初期组CD4~+、CD8~+T细胞频率在脾脏、肝脏、胸腺、腹股沟淋巴结中均显著增加(P<0. 05);与发病初期组比较,发病末期CD4~+T细胞频率在肝脏、胸腺、腹股沟淋巴结及外周血中均显著降低(P<0. 05)。②在脾脏、肝脏中,与未发病组和发病初期组比较,发病末期组Th1亚群比例显著增加(P<0. 05);在肝脏中,与发病初期组比较,发病末期组Th2、Th17亚群水平显著升高(P<0. 05)。③与未发病组比较,发病初期组肝脏、腹股沟淋巴结中iNKT细胞频率均显著增高(P<0. 05);与发病初期组比较,发病末期组外周血、肝脏中iNKT细胞频率显著降低(P<0. 05);与未发病组比较,发病初期组和发病末期组胸腺iNKT1亚群比例均显著增加,iNKT2亚群比例均显著降低(P<0. 05),脾脏、肝脏、腹股沟淋巴结iNKT1及iNKT2亚群比例三组两两比较均差异均无统计学意义(P>0. 05)。④在脾脏和腹股沟淋巴结中致炎性细胞因子和抑炎性细胞因子水平在发病初期较未发病组和发病末期组均显著升高(P<0. 05);在肝脏中致炎性细胞因子水平随小鼠病情进展逐渐升高,两两比较差异均有统计学意义(P<0. 05);抑炎性细胞因子水平在发病初期最高,发病末期显著降低(P<0. 05)。⑤胸腺PLZF相对表达量,三组两两比较差异均无统计学意义(P>0. 05);脾脏和肝,与未发病组和发病初期组比较,发病末期组T-bet相对表达量显著增加(P<0. 05)。结论:①发病初期CD4~+T和CD8~+T细胞的增加,特别是CD4~+T细胞的增加以及Th亚群的失衡是导致胰岛炎重要的免疫基础;②发病初期iNKT细胞频率的增加以及胸腺iNKT1/iNKT2亚群比例的翻转,提示了iNKT细胞在NOD/Ltj小鼠发病初期可能参与了T1D的发生。更多还原

【Abstract】 Objective: By observing and analyzing the CD4~+T,CD8~+T cells,Th1,Th2,Th17 subgroups,iNKT cell frequency and subsets,cytokines and related transcription factors of the NOD/Ltj mice in different tissues at the non-onset,early onset and late onset stage disease,to further understand the different stages of the cellular immune function of NOD/Ltj mice of type Ⅰdiabetes. Methods: The female NOD/Ltj mice were selected as the experimental subjects. The fasting blood glucose level of mice was detected by blood glucose meter and was based on urine glucose positive and twice consecutively ≥ 11. 1 mmol/L as the T1 D onset criteria. The mice were divided into non-onset group,early onset group and late onset group. Flow cytometry( FCM) was used to detect CD4~+T and CD8~+T cells,Th1,Th2,and Th17 subsets,and the frequency and subsets ratio of iNKT cells in the peripheral blood,thymus,spleen,liver of each group and CD4~+T and CD8~+T cells in the inguinal lymph nodes were also detected; IFN-γ,TNF-α,IL-2,IL-6,IL-17 A,IL-4,IL-10 were detected by Cytometric Bead Array( CBA); Western blot( WB) detected PLZF,T-bet,GATA-3,and ROR-γt. Results:(1)The frequency of CD4~+T and CD8~+T cells were significantly increased in the spleen,liver,thymus and inguinal lymph nodes in early onset group compared with the non-onset group( P<0. 05); late onset group compared with the early onset,the frequency of CD4~+T cells in liver,thymus,inguinal lymph nodes and peripheral blood were all significantly reduced( P < 0. 05).(2)In the spleen,liver,the ratio of Th1 subsets in the late onset group was significantly higher( P<0. 05) than that of the non-onset group and the early onset group. In the liver,the levels of Th2 and Th17 subset increased significantly in the late onset group( P<0. 05) compared with the early onset.(3)Compared with the non-onset group,the frequency of iNKT cells in the liver and inguinal lymph nodes of the early onset group was significantly higher( P < 0. 05); compared with the early onset group,the frequency of iNKT cells in peripheral blood and liver the of the late onset group was significantly lower( P < 0. 05); compared with the non-onset group,the proportion of iNKT1 subsets in the thymus of the early onset and the late onset group increased significantly,and the proportion of iNKT2 subsets decreased significantly( P < 0. 05),but the ratio of iNKT1 and iNKT2 in the spleen,liver and inguinal lymph nodes in the three groups was no significant difference between every two groups( P>0. 05).(4)The levels of inflammatory cytokines and anti-inflammatory cytokines in the spleen and inguinal lymph nodes were significantly higher in the early onset than in the non-onset and late onset groups( P<0. 05). The level of inflammatory cytokines in the liver increased gradually with the progression of the mice’s disease,there was a significant difference between every two groups( P<0. 05). The level of anti-inflammatory cytokines was highest in early onset group,but significantly decreased in late onset group( P < 0. 05).(5)The relative expression of PLZF in thymus was not significantly different between the three groups( P>0. 05).The relative expression of T-bet was significantly increased in the spleen and liver of late onset group,compared with the non-onset and early onset groups( P< 0. 05). Conclusion:(1)The increase of CD4~+T and CD8~+T cells,especially the increase of CD4~+T cells and the imbalance of Th subsets at the early stage of disease are the important immune basis for insulitis.(2)The increase of the frequency of iNKT cells and the reversal of the proportion of the thymus iNKT1/iNKT2 subgroup suggest that iNKT cells may participate in the occurrence of T1 D in the early onset of NOD/Ltj mice.更多还原