含转导域重组神经肽对D-半乳糖致免疫衰老小鼠胸腺功能的影响

Effect of restructured PACAP-PTD containing transduction domain’s on thymus function of D-galactos E-induced immunosenescence mice

目的探讨垂体腺苷酸环化酶激活肽(PACAP)及含蛋白转导域的重组PACAP(PACAP-PTD)对D-半乳糖(D-Gal)致免疫衰老小鼠胸腺功能的影响,明确其延缓免疫衰老的作用.方法将30只雄性BALB/c小鼠随机分组:对照组5只,每天颈背部皮下注射生理盐水(500 mg/kg);模型组25只,注射等量D-Gal溶液,连续注射42 d.再从模型组中随机选取18只均分为PACAP组、PACAP-PTD组、D-Gal组.PACAP组和PACAP-PTD组分别每天注射PACAP和PAC-AP-PTD溶液(12.5 ml/kg),D-Gal组注射等量的生理盐水、采用Real-time PCR法检测胸腺和脾脏初始T淋巴细胞标志物(sjTRECs)的含量以及胸腺相关基因T淋巴细胞易位蛋白(LMO2)、白细胞介素-7(IL-7)和叉头蛋白N1(Foxn1)的表达;流式细胞术检测全血中T细胞CD3、CD4、CD8的表型变化.结果小鼠胸腺和脾脏的初始T细胞中sjTRECs的含量:D-Gal组<PACAP组<PACAP-PTD组(P<0.01);与D-Gal组比较,PACAP和PACAP-PTD均能够上调免疫衰老小鼠显示胸腺功能和状态相关基因Foxn1、IL-7的表达水平(P<0.01),下调LMO2的表达水平(P<0.01),且PACAP-PTD比PACAP上调基因Foxn1、IL-7的表达水平更显著(P<0.01);小鼠全血T淋巴细胞亚群的CD3+CD4+/CD3+CD8+比值:D-Gal组<PACAP组<PACAP-PTD组(P<0.01).结论PACAP和PACAP-PTD显著增强小鼠胸腺初始T淋巴细胞生成和输出功能,维持外周T淋巴细胞受体库多样性;上调胸腺功能和状态相关基因的表达,扩展胸腺微环境;增强细胞免疫功能,恢复了退化的胸腺功能,具有延缓衰老和免疫保护作用.

Objective To explore the effects of pituitary adenylate cyclase activating polypeptide(PACAP)and recombinant PACAP(PACAP-PTD)containing protein transduction domain on thymus function in D-galactose(D-Gal)-induced immunosenescence mice,and to clarify its role in delaying immune aging.Methods Thirty male BALB/c mice were randomly divided into control group and model group,and 5 rats in the control group were injected subcutaneously with 500 mg/kg normal saline per day.The model group was injected with the same amount of D-Gal solution.After continuous injection for 42 days,18 rat in the model group were randomly selected as PACAP-PTD group,PACAP group and D-Gal group,6 for each group.The PACAP group and the PACAP-PTD group were intraperitoneally injected with 12.5 ml/kg PACAP and PACAP-PTD solution,and the D-Gal group was injected with the same amount of normal saline.Real-time PCR was used to detect initial T cell markers(sjTREC)in the thymus and spleen,the expression of the thymus-associated genes LIM domain only 2(LMO2),interleukin-7(IL-7)and forkhead box protein N1(Foxn1).Flow cytometry was used to detect the phenotypic changes of T lymphocytes CD3,CD4 and CD8 in whole blood of mice.Results The content of sjTRECs in T lymphocytes of mouse thymus and spleen:D-Gal group<PACAP group<PACAP-PTD group(P<0.01).Compared with the D-Gal group,PACAP and PACAP-PTD could up-regulate the expression of IL-7 and Foxn1(P<0.01),and down-regulate the expression of LMO2(P<0.01);moreover,PACAP-PTD up-regulated the expression of Foxn1 and IL-7 more significantly than PACAP(P<0.01).Ratio of CD3^+CD4^+/CD3^+CD8^+in mouse whole blood T lymphocyte subsets:D-Gal group<PACAP group<PACAP-PTD group(P<0.01).Conclusion PACAP and PACAP-PTD significantly enhance the initial T cell production and export function of mouse thymus,keep the diversity of peripheral T lymphocyte bank,up-regulate the expression of thymus function and statE-related genes,expand the thymus microenvironment,enhance cellular immune function and restore degraded thymic function,which have the effect of delaying aging and immune protection.