TIGIT及其配体CD155在“怒”应激大鼠胸腺细胞中的表达及其意义

Expression and significance of TIGIT and its ligand CD155 in thymocytes of rats under anger stress

目的探讨"怒"模型大鼠胸腺细胞中T细胞免疫球蛋白和ITIM结构域蛋白(TIGIT)及其配体CD155和血清促肾上腺皮质激素释放激素(CRH)、促肾上腺皮质激素(ACTH)、白细胞介素-2(IL-2)、白细胞介素-10(IL-10)表达情况,以及这些指标与胸腺细胞凋亡的相关性。方法将48只Wistar雄性大鼠随机分成对照组、模型7 d组、模型14 d组、模型21 d组,每组12只。除对照组外,其余各组以“社会隔离法”结合足底电击法复制精神应激“怒”模型大鼠。实验后测定大鼠体质量和胸腺指数,以苏木精-伊红(HE)染色观察大鼠胸腺组织的形态学改变,采用免疫印迹法检测各组大鼠胸腺细胞中TIGIT、CD155蛋白的表达水平,采用Tunel法检测胸腺细胞凋亡情况,采用酶联免疫吸附试验测定血清CRH、ACTH、IL-2、IL-10水平。结果实验后,4组大鼠体质量较试验前1 d升高(P<0.05),且“怒”模型各组大鼠体质量均低于对照组(P<0.05);实验后“怒”模型各组大鼠胸腺指数均低于对照组(P<0.05)。HE染色光学显微镜下(×400)可见“怒”模型各组大鼠大鼠胸腺皮质组织结构出现不同程度的损坏,幼稚T细胞布排紊乱无序或呈“条索状”堆积,细胞间隙缩小,细胞数量较对照组降低,细胞核染色变浅,呈淡紫色,并出现不同程度核固缩、核分裂、细胞溶解等凋亡细胞增多现象。4组大鼠胸腺细胞TIGIT蛋白表达水平比较,对照组<模型21 d组<模型14 d组<模型7 d组,其中模型7 d组、14 d组与其他各组胸腺细胞TIGIT蛋白表达水平差异均有统计学意义(P<0.05);“怒”模型各组大鼠胸腺细胞CD155蛋白表达水平高于对照组,差异均有统计学意义(P<0.05)。“怒”模型各组胸腺组织凋亡细胞吸光度高于对照组,差异有统计学意义(P<0.05)。模型7 d组、模型14 d组血清CRH、ACTH水平高于对照组及模型21 d组(P<0.05);“怒”模型各组IL-2水平低于对照组(P<0.05),且模型7 d组、模型14 d组低于模型21 d组(P<0.05);“怒”模型各组IL-10水平高于对照组(P<0.05),且模型14 d组高于模型7 d组及模型21 d组(P<0.05)。Pearson相关分析结果显示,TIGIT表达水平与CD155、ACTH、CRH及胸腺细胞凋亡程度呈正相关(P<0.05),与IL-2呈负相关(P<0.05),与IL-10及胸腺指数无相关性(P>0.05);CD155表达水平与TIGIT及胸腺细胞凋亡程度呈正相关(P<0.05),与IL-2呈负相关(P<0.05),与ACTH、CRH、IL-10及胸腺指数无相关性(P>0.05)。结论“怒”应激状态下,胸腺出现萎缩及凋亡细胞增多现象,可能是TIGIT-CD155与下丘脑-垂体-肾上腺轴、IL-10、IL-2共同作用所致,该研究为“怒”应激致使免疫损伤及疾病发生发展提供理论依据。

Objective To investigate the expression of T cell immunoglobulin and ITIM domain protein(TIGIT)and its ligand CD155 in thymocytes of rats under"anger"model and the expression of serum corticotropin releasing hormone(CRH),adrenocorticotropic hormone(ACTH),interleukin-2(IL-2)and interleukin-10(IL-10),as well as the correlation between these indicators and thymocyte apoptosis.Methods Forty-eight Wistar male rats were randomly divided into control group,model 7 d group,model 14 d group and model 21 d group,with 12 rats in each group.Except the control group,the mental stress"anger"model rats in the other groups were replicated by the"social isolation method"combined with the plantar electric shock method.After the experiment,the body mass and thymus indicators of rats were measured,the morphological changes of thymus tissue were observed by hematoxylin-eosin(HE)staining,the expression levels of TIGIT and CD155 protein in thymocytes of rats in each group were detected by Western blotting,the apoptosis of thymocytes was detected by the Tunel method,and the levels of serum CRH,ACTH,IL-2 and IL-10 were measured by enzyme-linked immunosorbent assay.Results After the experiment,the body mass of rats in the four groups increased compared with the day before the experiment(P<0.05),and the body mass of rats in the"anger"model group was lower than that in the control group(P<0.05);after the experiment,the thymus index of"anger"model group was lower than that of the control group(P<0.05).Under the HE staining optical microscope(×400),the thymic cortex structure of rats in each group of the"anger"model showed different degrees of structural damage,the arrangement of immature T cells was disordered or stacked in a"string"shape,and the intercellular space was narrowed,the number of cells decreased compared with the control group,the nucleus staining was lighter and light purple,and the apoptotic cells increased in varying degrees,such as nuclear pyknosis,mitosis and cytolysis.The comparison result of expression levels of TIGIT protein in thymocytes of the four groups is the control group<the model 21 d group<the model 14 d group<the model 7 d group,and there were significant differences in the expression levels of TIGIT protein among the model 7 d group and model 14 d group and other groups(P<0.05).The expression level of CD155 protein in thymocytes of rats in the"anger"model group was higher than that in the control group,and the differences was statistically significant(P<0.05).The absorbance of apoptotic cells in thymus tissue of the"anger"model group was higher than that of control group,and the difference were statistically significant(P<0.05).The levels of serum CRH and ACTH in model 7 d group and model 14 d group were higher than those in control group and model 21 d group(P<0.05);the level of IL-2 in the"anger"model group was lower than that in control group(P<0.05),and the level of IL-2 in Model 7 d group and model 14 d group was lower than that in model 21 d group(P<0.05);the level of IL-10 in the"anger"model group was higher than that in the control group(P<0.05),and the level of IL-10 in the model 14 d group was higher than that in the model 7 d group and model 21 d group(P<0.05).Pearson correlation analysis results showed that TIGIT expression level was positively correlated with CD155,ACTH,CRH and thymocyte apoptosis(P<0.05),negatively correlated with IL-2(P<0.05),and had no correlation with IL-10 and thymus index(P>0.05).CD155 expression level is positively correlated with TIGIT and thymocyte apoptosis(P<0.05),negatively correlated with IL-2(P<0.05),and had no correlation with ACTH,CRH,IL-10 and thymus index(P>0.05).Conclusion Under the"anger"stress state,thymus atrophy and apoptotic cells increase,this research may be caused by the joint action of TIGIT-CD155 with the hypothalamic-pituitary-adrenal axis,IL-10 and IL-2,which provides a theoretical basis for immune injury and disease occurrence and development caused by"anger"stress.