补肾健脾方干预PPD诱导小鼠T细胞耗竭的实验研究

摘要

Objective:To evaluate effects of Bushen Jianpi Recipe ( BSJP) on restoring of T cell exhaustion induced by persistent tuberculosis antigens in mice.Methods:The C57BL/6 mice were primed with BCG(5×106 CFU)and boosted with 20μg of Mycobacterium bovis BCG Purified Protein Derivative ( PPD ) once a week and lasted for 16 times to induce T cell exhaustion that happened in late stage of tuberculosis .BSJP was used to treat the mice with immune exhaustion for 3 weeks.Flow Cytometry was used to detect the numbers of CD 4+and CD8+T cells and PD-1 expression.ELISA was used to detect IL-2 and IFN-γsecretion.Bacteria load in lung tissue following BCG challeng was used to detect the protective capability of mice models .Results: Compared with the transient antigen stimulation group,the persistent antigen stimulation group had lower level of IFN-γand IL-2 secretions(P<0.01).The numbers of CD4+and CD8+T cells decreased and the level of inhibitory receptor PD-1 got higher(P<0.01).The protective efficacy against BCG challenging decreased ( P<0.01 ) .After treatment with BSJP , the production of cytokines IFN-γand IL-2 increased ( P<0.01 );the numbers of CD4+and CD8+T cells increased and PD-1 expression level on CD4+T cells decreased(P<0.01);the protective efficacy against BCG challenge increased (P<0.01).The results showed that BSJP can reduce the PD-1 expression,improve the IL-2 and IFN-γsecretion,and increase the protection against BCG challenge .Conclusion:Bushen Jianpi Recipe can restore T cell exhaustion induced by persistent tuberculosis antigen stimulation in mice , which gives a hope to overcome T cell exhaustion in tuberculosis with Chinese herbal in clinic.%目的:研究结核分枝杆菌抗原持续刺激致T细胞功能耗竭的机制,探究补肾健脾方对T细胞功能耗竭的治疗作用.方法:利用BCG初免,结核菌素纯蛋白衍化物(PPD)持续刺激C57BL/6小鼠,模拟结核分枝杆菌抗原在病人体内持续存在所造成的慢性感染病理过程,构建T细胞耗竭模型.在T细胞耗竭模型基础上,给予补肾健脾方灌胃治疗.治疗3周后检测不同实验组细胞免疫水平,包括利用流式细胞术检测脾脏CD4+和CD8+T细胞数目,以及细胞表面抑制性受体PD-1的表达水平;利用ELISA检测脾脏淋巴细胞分泌IL-2和IFN-γ的水平;利用CFU菌落计数检测肺脏BCG攻击后的抗感染免疫能力.结果:与抗原短暂刺激组相比,免疫耗竭未治疗组CD4+和CD8+T细胞数目显著下降(P<0.05),细胞表面PD-1表达水平显著增高(P<0.01);脾淋巴细胞分泌IL-2和IFN-γ明显降低(P<0.01);小鼠经BCG攻击后,抗感染保护效果显著下降(P<0.01).经补肾健脾方治疗后,脾脏CD4+和CD8+T细胞数目得到明显恢复(P<0.05),CD4+T细胞PD-1的表达得到显著降低(P<0.01);脾淋巴细胞分泌IL-2和IFN-γ的水平明显增高(P<0.05);小鼠抗感染的能力显著性增强(P<0.01).结论:通过结核抗原PPD持续刺激成功构建T细胞耗竭模型;补肾健脾方能逆转抗原持续刺激导致的小鼠T细胞功能耗竭,可为临床治疗肺结核T细胞功能耗竭提供参考.