慢性肾衰大鼠Th17/Treg细胞内质网应激及枸橼酸对其影响

The various complications caused by the immunologic derangement are the main reasons of of death in patients with chronic renal failure. The research found that hemodialysis patients have Th17 cells increased and Treg cells decreased. The imbalance of Th17/Treg cells play an important role in their immunologic derangement, but the extent and mechanisms of this imbalance in several stages of renal failure patients are not clear. Patients of Chronic renal failure have severe endoplasmic reticulum stress (ERS), ERS is involved in the regulation of immune cells. We research ERS in Th17/Treg cells that may help us to clarify its imbalance mechanism. Early study found that citric acid as an antioxidant can improve cellular immune function. This project will establish the rat model of chronic renal failure, then use of flow cytometry, cell culture and molecular biology methods to study. Firstly, we study Th17/Treg cell number and function of imbalance mechanism in the different stages of renal failure; Secondly, we study the relationship between PERK, ATF6, IRE1/XBP1 signal pathway or the specific apoptotic transcription factor GADD153 related to the ERS and the imbalance of Th17/Treg cells. Lastly, we blocked the GADD153, ERS specific apoptotic pathway, with RNA interference technology and observed effects of citrate to Th17/Treg cells. This project may help to learn the regular pattern and the mechanisms of imbalance of Th17/Treg cells in chronic renal failure, and provide a theoretical basis to look for new drug which can prevent and treat immunologic derangement and prolong life of patients of chronic renal failure.

免疫紊乱导致的各种并发症是慢性肾衰患者的主要死亡原因。前期发现血透患者Th17细胞上升,而Treg细胞下降。Th17/Treg细胞失衡确实在其免疫紊乱中占重要地位,但肾衰不同阶段Th17/Treg细胞失衡的程度和机制目前尚不清楚。慢性肾衰患者存在严重的内质网应激（ERS），而ERS参与了免疫细胞的调控,对Th17/Treg细胞ERS的研究有助于阐明其失衡机制。前期发现枸橼酸作为抗氧化剂具有改善细胞免疫作用。本课题建立慢性肾衰大鼠模型,利用流式细胞仪、细胞培养及分子生物学方法,研究肾衰不同阶段Th17/Treg细胞数量及功能失衡规律;ERS相关的PERK、ATF6、IRE1/XBP1信号通路及特异凋亡转录因子GADD153与Th17/Treg细胞失衡的关系;RNA干扰GADD153阻断ERS特异凋亡途径,观察枸橼酸对Th17/Treg细胞的影响。为寻找防治慢性肾衰免疫紊乱的药物提供理论依据。

背景：内质网应激是细胞应对有害刺激导致的各种疾病（包括慢性肾衰竭）时的重要自身保护机制。枸橼酸钠具有抗氧化、改善细胞免疫等作用，通常被用于慢性肾衰透析时的抗凝剂和改善慢性肾衰竭病人的免疫紊乱。但对于枸橼酸钠是否具有抗内质网应激、改善慢性肾衰竭肾脏组织内质网应激目前尚不清楚。调节性T细胞( Treg) 和辅助性T细胞17(Th17)在维持机体的免疫平衡方面发挥着非常重要的作用。近来研究表明在维持性血液透析患者有明显的Th17/Treg功能失衡，表现为Th17细胞数目上升，而Treg细胞数目下降，慢性肾衰Th17/Treg细胞的内质网应激是否是引起细胞失衡的机制之一?对其进一步研究可在调节Th17/Treg细胞平衡靶点对慢性肾衰免疫紊乱的防治提供新途径。.研究内容：1.研究慢肾衰大鼠体内是否存在内质网应激和Th17/Treg细胞失衡，以及在枸橼酸干预后是否可以改善免疫紊乱状态。2.检测慢性肾衰竭患者不同发展阶段内质网应激相关指标GRP78、CHOP与Th17/Treg 细胞的变化趋势，分析在不同慢性肾脏病发展阶段内质网应激与免疫状态的规律性。3.体外研究内质网应激对Th17/Treg细胞的影响：分离大鼠脾脏单核细胞，内质网应激诱导剂毒胡萝卜素刺激及枸橼酸干预后，Th17/Treg细胞数量及分泌相应细胞因子的变化。.重要结果：1.枸橼酸钠可以通过改善内质网应激对慢肾衰大鼠起保护作用；枸橼酸钠在较高剂量时对慢性肾衰竭大鼠的肾脏保护作用较强。.2.枸橼酸钠可以降低慢肾衰大鼠Th17细胞的活性及其相关细胞因子的水平，提高Treg细胞的活性及其相关细胞因子的水平；降低慢肾衰大鼠Th17/Treg的比值。.3.随着慢性肾衰竭患者病程进展Th17及Treg细胞变化逐渐明显，且两者的变化并不同步，导致了Th17/Treg比值的失衡逐渐加重，失衡状态始终贯穿于疾病进展过程，Th17/Treg细胞比值失衡与内质网应激反应呈正相关，可能是内质网应激导致了Th17/Treg细胞的失衡。.4.在体外内质网应激诱导剂毒胡萝卜素会造成Th17/Treg比值失衡，枸橼酸钠可以改善这一失衡状态。.科学意义：为寻找防治慢性肾衰免疫紊乱的药物提供理论依据。