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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。
OBJECTIVE: To establish an ELISPOT assay for detecting Th1 / Th2 lymphocyte subsets in splenic lymphocytes of rats and further compare the effects of Th1 / Th2 lymphocytes and CD4 + / CD8 + The process of change, in order to screen the best immunological evaluation indicators provide a reference. Methods: Female SD rats were subjected to 9-week incremental treadmill training. Rats were sacrificed at 36 hours after training in the first week, the third week and the ninth week of training respectively, and one week after the recovery. Different kinds of stimulants and different stimulant concentrations were used to stimulate different numbers of splenic lymphocytes of rats to establish the best ELISPOT assay. The levels of hemoglobin, testosterone and corticosterone in rats were measured to establish a model of fatigue. Th1 / Th2 lymphocyte subsets and CD4 + / CD8 +, a traditional immunological marker, were dynamically observed. RESULTS: The best ELISPOT assay was established: PMA and Ionomycin combined. The concentration of PMA was 2.5 ng / ml when detecting interferon-γ (IFN-γ) and 10 ng / ml when detecting interleukin-4 (IL-4). The concentration of Ionomycin was 0.05 μg / ml, Ionomycin concentration was 0.2μg / ml when IL-4 was detected. Cell concentration: 3 × 10 4 cells / well (IFN-γ), 60 × 10 4 cells / well (IL-4). 36 hours after exercise, exercise group hemoglobin, testosterone and corticosterone were significantly decreased compared with the control group; after 1 week recovery, compared with the control group no significant difference. Compared with the control group, Th1-type cytokine IFN-γ in exercise group increased significantly after treadmill training in the first week of exercise. Compared with control group, treadmill training decreased significantly after treadmill training in the third week; After exercise, exercise group IFN-γ significantly decreased compared with the control group. During the recovery period after the incremental load training, the level of Th2 cytokine IL-4 in exercise group was 1.44 times higher than that in control group (P <0.01). No significant changes in CD4 + / CD8 + during the whole training period and recovery period. CONCLUSION: Compared with CD4 + / CD8 +, ELISPOT is more sensitive to detect the changes of Th1 / Th2 lymphocyte subsets in SD rats during incremental over-training.