固相酶联免疫斑点技术检测递增负荷过度训练大鼠细胞免疫机能变化研究

目的:建立大鼠脾脏淋巴细胞中Th1/Th2型淋巴细胞亚群表达的固相酶联免疫斑点(ELISPOT)技术检测方案,进一步比较Th1/Th2淋巴细胞和CD4+/CD8+在大鼠递增负荷过度训练过程中的改变,为筛选最佳免疫学评价指标提供参考。方法:雌性SD大鼠进行9周递增负荷跑台训练,分别在训练的第1周、第3周、第9周训练结束后的36小时,以及恢复1周后处死大鼠。采用不同种类刺激剂、不同刺激剂浓度刺激不同数量大鼠脾脏淋巴细胞,建立最佳ELISPOT检测方案。检测大鼠血红蛋白、睾酮、皮质酮,评估疲劳模型的建立,并对Th1/Th2型淋巴细胞亚群以及传统免疫学指标CD4+/CD8+进行动态观察。结果:建立最佳ELISPOT技术检测方案:PMA和Ionomycin联合刺激。刺激物浓度:检测γ干扰素(IFN-γ)时PMA浓度为2.5ng/ml,检测白细胞介素4(IL-4)时PMA浓度为10ng/ml;检测IFN-γ时Ionomycin浓度为0.05μg/ml,检测IL-4时Ionomycin浓度为0.2μg/ml。细胞浓度:3×104cells/孔(IFN-γ),60×104cells/孔(IL-4)。运动后36小时,运动组血红蛋白、睾酮、皮质酮均较对照组显著下降;经1周恢复后,和对照组相比无显著性差异。递增负荷运动第1周跑台训练后,运动组Th1型细胞因子IFN-γ与对照组相比显著升高;第3周跑台训练后,运动组和对照组相比显著下降;恢复1周后,运动组IFN-γ与对照组相比显著下降。递增负荷训练结束后的恢复期,运动组Th2型细胞因子IL-4较对照组升高了1.44倍(P<0.01)。CD4+/CD8+在整个训练期及恢复期均无显著性变化。结论:递增负荷过度训练过程中,与CD4+/CD8+相比,采用ELISPOT技术检测SD大鼠Th1/Th2淋巴细胞亚群的改变具有较高的灵敏性。

ELISPOT Detection of Cell Immunity in Over-Loaded Rats with Incremental Exercise

Aim To develop the operating procedure of enzyme-linked immunodotting spot(ELISPOT) for detecting the changes in Th1/Th2 of over-loaded SD rats with incremental exercise. Methods Healthy female SD rats were trained on a motor-driven treadmill with a progressive incremental load for 9 weeks. Samples were collected at: 36 hours post-training after 1st week,3rd week and 9th week,and 7 days after conclusion of training. Establishment of the ELISPOT operating procedure was based on the different stimulants and their different concentrations,and different numbers of spleen lymphocytes. Comparison was done between dynamic changes of Th1/Th2 and CD4+/CD8+. Results The best ELISPOT detecting procedure included combined stimulation of PMA(IFN-γ: 2.5ng/ml; IL-4: 10ng/ml) and Ionomycin(IFN-γ: 0.05 μg/ml; IL-4: 0.2 μg/ml),and cells concentration(3 ×104 cells/well for interferon-γ and 60 ×104 cells/well for IL-4). The levels of Hb,plasma T and C 36 hours after exercise were significantly lower in exercise group than that in control group. Th1 cytokine IFN-γ increased after the first week training and decreased after the 3rd week training as compared with the control group. IFN-γ in exercise group sharply declined at 7th day after the conclusion of training as compared with the control group. The expression of Th2 cytokine IL-4 7 days after exercise in exercise group was 1.44 folds higher than that in control group(P <0.01). The ratio of CD4+T/CD8+T changed insignificantly throughout the whole training and recovery periods. Conclusion Utilization of ELISPOT for detecting the change in Th1/Th2 lymphocytes subsets appeared to be more sensitive than the traditional determination of CD4+/CD8+ ratio in overloaded rats with incremental exercise.