慢性移植性肾病大鼠脾脏中辅助性T细胞和B细胞免疫状态的研究

The Immune Status of Th and B Cells in the Rat Spleen during Chronic Allograft Nephropathy

目的:检测慢性移植性肾病(CAN)大鼠脾脏中辅助性T细胞(Th)和B细胞特征性因子表达量的变化,探究这些Th/B细胞免疫状态在CAN病程中的作用。方法:采用Fischer-Lewis左肾原位移植法建立大鼠慢性移植性肾病模型,Lewis-Lewis同种自体移植作为对照组。所有受体大鼠,术后8周处死,取脾脏组织,进行HE染色,拍照后采用双盲法评价脾脏组织病理变化程度及淋巴细胞浸润情况。用Trizol法提取脾脏组织中总RNA,采用实时荧光定量聚合酶链式反应(q RT-PCR)法检测各组脾脏中Th1,Th2,Th17,Treg和B细胞标志性因子的表达情况。结果:与对照组相比,CAN大鼠脾脏出现明显的结构肿胀及淋巴细胞浸润增多,并且Th1细胞特征性因子IFN-γ和T-bet表达量显著增加(P<0.001,P<0.05);Th2细胞特征性因子GATA-3表达升高(P<0.001),但IL-4无变化;IFN-γ/IL-4比例明显上调(P<0.001),T-bet/GATA3比例没有显著差异。Th17的特征性因子IL-17未见明显改变,而Treg细胞特征性因子Foxp3表达增加(P<0.001),IL-17/Foxp3平衡明显向Treg细胞偏移(P<0.05)。B细胞激活相关因子TNFRSF13C和RAG1表达量均显著上调(P<0.01,P<0.05),而RAG2水平则没有变化。结论:CAN大鼠脾脏中Th1/Th2的活性平衡向Th1偏移,分化平衡未出现显著变化;Th17/Treg的平衡向Treg细胞偏移,B细胞免疫状态也被激活,这些变化在CAN病程的发展中起到了重要作用,并且为临床监测和治疗提供了新的依据。

Objective： To explore the immune statuses of splenic Th and B cells in a chronic allograft nephropathy（CAN） rat model, and initially investigate the potential role of them in CAN. Methods： Chronic allograft nephropathy rat model was established by orthotopic renal transplantation from Fischer to Lewis rats. Lewis to Lewis as the controls. Spleen samples were obtained 8 weeks after transplantation. Sections of spleen tissues were stained by hematoxylin-eosin（HE）, and the histopathology changes were elvaluated a double-blind review. Besides, the expression of Th1/Th2/Th17/Treg/B-cell-associated immunological molecules were examined byquantitative Real-time PCR（q RT-PCR）. Results： There were splenic nodules swelling and a significant increase of lymphocyte infiltration in the CAN group as compared to NC group. The expression of Th1-related molecules（IFN-γ and T-bet） were increased（P〈0.001, P〈0.05）, while only Th2-related transcription factor GATA3 not IL-4 was enhanced（P〈0.001）. Similarly, IFN-γ/IL-4 rather than T-bet/GATA3 was up-regulated in the spleen with CAN（P〈0.001）. Moreover, there were parallel Th17-related cytokines IL-17 level but higher Foxp3 level of Treg（P〈0.001）, as well as lower IL-17/Foxp3（P〈0.05） in the CAN rats than NC individuals. Besides, the expression of TNFRSF13 C and RAG1 which involving in B cell activation were also improved in CAN recipients（P〈0.01,P〈0.05）.Conclusions： The changes of these Th and B cell immune statuses in the spleen are closely associated with the pathological process of CAN, which also provides a novel clue for monitoring this disease.