HMGB1在肾脏热、冷缺血再灌注损伤中作用机理及干预研究

发布时间：2018-09-04 08:41

【摘要】：正文：尿毒症为慢性肾功能不全的终末阶段,在我国每年新发病率为0.01%,其中大多数为青壮年,严重影响了人们的健康和生命安全。肾移植是治疗终末期肾病的最佳治疗手段,可以显著的提高终末期肾病患者的生活质量,减少并发症的发生,并降低终末期肾病的死亡率。虽然随着移植手术技巧的改进,新型免疫抑制药物的应用,围手术期并发症及早期急性排斥反应发生率明显减低,大大增加了移植肾一年的存活率,然而移植肾的半寿期并未明显提高,慢性移植肾病受到越来越多的关注。移植肾冷缺血所造成的冷缺血再灌注损伤是临床器官移植不可避免的病理生理过程,是慢性移植肾病的危险因素之一。供肾的冷缺血时间(coldischemia time CIT)是造成肾移植患者术后发生移植肾功能延迟恢复(delayed graft function DGF)的主要原因,而DGF和急性排斥反应(acute rejection episodes ARE)的发生是影响肾脏存活时间的重要因素近年来的研究发现,炎症、免疫反应参与了缺血再灌注损伤。缺血再灌注过程中炎症细胞聚集增加、细胞因子表达上调及T细胞介导的免疫反应等是造成肾组织损伤的重要因素。免疫系统是机体在长期进化中逐步形成的一套机制复杂的防御系统,包括天然免疫和获得性免疫。现已广泛认为,获得性免疫的激活需要先天性免疫系统的参与,尤其是先天性免疫系统中的TLRs家族,它们是先天性免疫的信号发动受体,TLRs除了识别病原相关的分子模式(PAMP),如细菌、病毒、真菌和原虫外,同样能识别多种因素引起损伤时受损细胞或坏死细胞释放的内源性配体,如高迁移率族蛋白B1(high mobility group box 1,HMGB1)等,进而引发一系列的炎症反应. HMGB1是一类早在30多年前就发现的非组蛋白(nonhistone chromatin protein,NHCP),属于高迁移率族蛋白(high mobility group protein,HMG)家族,早期的研究认为HMGB1是一种核内蛋白,通过与DNA的相互作用参与核内诸如转录、复制、重组等复杂有序的功能过程。近年来的研究发现,核外HMGB1可能具有“早期”和“晚期”炎症介质的作用,在关节炎、脓毒血症、创伤、急性肺损伤、缺血再灌注损伤等各类急慢性炎疾病的中,HMGB1被证实扮演了重要的角色。在目前国内及国外的研究中,针对HMGB1的研究集中在以大鼠热缺血再灌注为模型的动物实验。研究提示内源性配体(HMGB1)与TLRs结合能诱导树突状细胞(DC)的成熟,除了产生细胞因子和化学因子,成熟的DC表面共同刺激分子能提供“第二信号”给T淋巴细胞。TLRs与配体结合后在DC中通过T辅助细胞(TH细胞)激活来引发效应。反之,若缺乏TLRs的外源性和内源性配体,那么TLRs无法激活,则DC成熟障碍,而未成熟的DC不能启动T淋巴细胞反应。肾脏冷缺血再灌注损伤机制、损伤特点尚不明确。实验研究中,阻断胞外HMGB1的活性,已成为干预相关疾病过程的重要策略。丙酮酸乙酯(Ethyl pyruvate,EP)是美国食品和药品管理局划分为无毒性物质的一种食品添加剂,有显著的抑制HMGB1的合成与释放作用,对器官缺血再灌注损伤有保护作用,对其进行进一步研究具有重大的临床价值。本课题通过研究HMGB1在不同大鼠肾脏缺血再灌注模型中的表达,以及应用EP进行干预的结果,旨在讨论如下问题：1、构建大鼠肾脏热缺血再灌注损伤及冷缺血再灌注损伤,观察缺血后不同时间段HMGB1,炎症因子NF-κB、TNF-α的表达,以及肾脏损伤程度；2、探讨HMGB1启动肾脏热、冷血再灌注损伤后获得性免疫反应的机制；3、对于大鼠热缺血再灌注损伤进行EP干预性治疗,并观察疗效,并据此进行大鼠冷缺血再灌注损伤治疗,以提供防治肾脏冷缺血再灌注损伤的免疫学治疗方案。在目前器官供体紧缺情况下,如何维持器官冷缺血时间更长的保存期,以拓宽供体的使用范围,减少因供体冷缺血时间过长而导致移植后的移植物功能延迟恢复及移植器官病的发生率,延长移植肾半衰期,有重要的研究意义。第一章HMGB1在热缺血再灌注损伤中肾脏表达和干预研究目的：探讨HMGB1引发肾脏热缺血再灌注损伤的途径,明确HMGB1在热缺血再灌注损伤中肾脏表达,应用EP进行干预性治疗,明确EP进行治疗的效果。方法：60只健康清洁级Sprague-Dawley (SD)雄性大鼠被随机分为假手术组(n=20)、热缺血再灌注组(n=20)、丙酮酸乙酯(EP)治疗组(n=20)。采用夹闭左肾蒂使左侧肾脏缺血45 min再恢复血流并切除右侧肾脏制模。EP治疗组缺血前20min及再灌注前分别经阴茎背静脉注射EP 3.26 g/L(溶于林格液1 ml中),假手术组和热缺血再灌注组均经阴茎背静脉分别注射林格液1 ml,于术后6h、1d、3d、5d取下腔静脉血检测血肌酐、尿素氮水平,处死动物后取肾脏组织标本,应用RT-QPCR方法检测NF-κB、肿瘤坏死因子α(TNF-α),应用western blot方法检测HMGB1表达及免疫组化方法检测肾脏损伤程度。结果：热缺血再灌注组及EP治疗组HGMB1水平均高于正常对照组,其中热缺血再灌注组高于EP治疗组；热缺血再灌注组和EP治疗组血清CRE、BUN水平均显著高于正常对照组,其中热缺血再灌注组高于EP治疗组；热缺血再灌注组和EP治疗组NF-κB、TNF-α水平均显著高于正常对照组,其中热缺血再灌注组高于EP治疗组；热缺血再灌注组及EP治疗组肾脏损伤程度。结论：各组肾脏标本中均检测到HMGB1的表达,肾脏热缺血再灌注损伤后HMGB1表达升高,导致CRE、BUN、TNF-α、NF-κB表达增高,应用EP可减少HMGB1表达,CRE、BUN、TNF-α、NF-κB升高程度减低,有助于减轻热缺血再灌注所致肾损伤。第二章HMGB1在冷缺血再灌注损伤中肾脏表达和干预治疗目的：探讨HMGB1引发肾脏冷缺血再灌注损伤的机制,明确HMGB1在肾脏冷缺血再灌注损伤中的表达。根据热缺血再灌注损伤试验结果,应用EP进行干预性治疗,明确EP进行治疗的效果。方法：60只健康清洁级Sprague-Dawley(SD)雄性大鼠被随机分为假手术组、冷缺血再灌注组、丙酮酸乙酯(EP)治疗组。构建右肾冷缺血再灌注损伤模型。EP治疗组缺血前20min及再灌注前分别经阴茎背静脉注射EP 3.26 g/L(溶于林格液1 ml中),假手术组和冷缺血再灌注组均经阴茎背静脉分别注射林格液1 ml,于术后6h、1d、3d、5d取下腔静脉血检测血肌酐、尿素氮水平,应用RT-QPCR方法检测NF-κB、肿瘤坏死因子α(TNF-α),处死动物后取肾脏组织标本,应用western blot及免疫组化方法检测肾组织HMGB1表达情况及肾脏损伤程度。结果：冷缺血再灌注组及EP治疗组HGMB1水平均高于正常对照组,其中冷缺血再灌注组高于EP治疗组；冷缺血再灌注组和EP治疗组血CRE、BUN水平均显著高于正常对照组,其中冷缺血再灌注组高于EP治疗组；冷缺血再灌注组和EP治疗组NF-κB、TNFα水平均显著高于正常对照组,其中冷缺血再灌注组高于EP治疗组。结论：各组肾脏标本中均检测到HMGB1的表达,肾脏冷缺血再灌注损伤HMGB1、CRE、BUN、TNF-α、NF-κB变化趋势与热缺血再灌注损伤相同,但升高程度较低。考虑损伤机制与热缺血再灌注损伤相同,应用EP阻断HMGB1有助于减轻冷缺血再灌注所致肾损伤。
[Abstract]:Main text: Uremia is the end stage of chronic renal insufficiency, the new incidence rate is 0.01% every year in China, most of which are young adults, seriously affecting people's health and life safety. Kidney transplantation is the best treatment for end-stage renal disease, can significantly improve the quality of life of patients with end-stage renal disease, reduce complications. Although the incidence of perioperative complications and early acute rejection was significantly reduced with the improvement of transplantation techniques and the use of new immunosuppressive drugs, the one-year survival rate of transplanted kidneys was greatly increased. However, the half-life of transplanted kidneys was not significantly improved and chronic transplanted kidney disease was suffered. More and more attention.
Cold ischemia-reperfusion injury caused by cold ischemia is an inevitable pathophysiological process in clinical organ transplantation, and is one of the risk factors of chronic kidney transplantation. DGF and acute rejection episodes ARE are important factors affecting renal survival
In recent years, it has been found that inflammation and immune response are involved in ischemia-reperfusion injury. Increased aggregation of inflammatory cells, up-regulation of cytokine expression and T cell mediated immune response are important factors causing renal tissue injury during ischemia-reperfusion.
The immune system is a complex set of defense systems, including innate immunity and acquired immunity. It is widely believed that the activation of acquired immunity requires the involvement of innate immune system, especially the TLRs family in the innate immune system, which are signal-triggering receptors of innate immunity. In addition to recognizing pathogen-related molecular patterns (PAMPs), such as bacteria, viruses, fungi and protozoa, LRs can also identify endogenous ligands released by damaged or necrotic cells caused by various factors, such as high mobility group box 1 (HMGB1), which triggers a series of inflammatory reactions.
HMGB1 is a kind of non-histone chromatin protein (NHCP) discovered more than 30 years ago. It belongs to the high mobility group protein (HMG) family. Early studies suggested that HMGB1 is a kind of intranuclear protein, which participates in complex and orderly functions such as transcription, replication and recombination through the interaction with DNA. Recent studies have found that extranuclear HMGB1 may play an "early" and "late" role in inflammatory mediators. HMGB1 has been proved to play an important role in various acute and chronic inflammatory diseases such as arthritis, sepsis, trauma, acute lung injury, ischemia-reperfusion injury.
At present, studies on HMGB1 have focused on animal models of warm ischemia-reperfusion injury in rats. It is suggested that the binding of endogenous ligand (HMGB1) with TLRs can induce the maturation of dendritic cells (DC). In addition to the production of cytokines and chemical factors, the co-stimulatory molecules on the surface of mature DC can provide a "second message". TLRs bind to ligands and activate T helper cells (TH cells) in DCs to trigger the effect. Conversely, if TLRs are absent from the endogenous and exogenous ligands, then TLRs can not activate, then DC maturation is impaired, while immature DCs can not initiate T lymphocyte response. Vague.
Ethyl pyruvate (EP) is a kind of food additive classified as non-toxic substance by the Food and Drug Administration of the United States, which can significantly inhibit the synthesis and release of HMGB1 and protect organs from ischemia-reperfusion injury. It is of great clinical value to further study it.
The purpose of this study was to investigate the expression of HMGB1 in different rat models of renal ischemia-reperfusion and the results of EP intervention. The following questions were discussed: 1. To construct rat kidneys with warm ischemia-reperfusion injury and cold ischemia-reperfusion injury, to observe the expression of HMGB1, inflammatory factors NF-kappa B, TNF-alpha at different time intervals after ischemia, and to observe the expression of HMGB1, NF-kappa Secondly, to explore the mechanism of acquired immune response after cold blood reperfusion injury; thirdly, to observe the therapeutic effect of EP intervention on warm ischemia reperfusion injury in rats, and to treat cold ischemia reperfusion injury in rats, so as to provide immunological therapy for preventing and treating cold ischemia reperfusion injury in kidney. In the current situation of organ donor shortage, it is of great significance to study how to maintain a longer shelf life of cold ischemia in order to broaden the scope of use of donors, reduce the incidence of delayed graft function recovery and organ disease after transplantation due to long cold ischemia of donors, and prolong the half-life of transplanted kidneys.
Chapter 1 HMGB1 expression and intervention in renal ischemia / reperfusion injury
Objective: To explore the pathway of renal warm ischemia-reperfusion injury induced by HMGB1, to clarify the expression of HMGB1 in the kidney during warm ischemia-reperfusion injury, to apply EP intervention therapy, and to clarify the effect of EP treatment. Methods: 60 healthy clean Sprague-Dawley (SD) male rats were randomly divided into sham operation group (n=20), warm ischemia-reperfusion group (n=2). Ethyl pyruvate (EP) treatment group (n=20). The left kidney ischemia was restored for 45 minutes by clipping the left kidney pedicle and the right kidney was resected. EP 3.26 g/L (dissolved in Ringer's solution 1 ml) was injected into the dorsal penile vein 20 minutes before ischemia and before reperfusion in the EP treatment group, and EP 3.26 g/L (dissolved in Ringer's solution 1 ml) was injected through the dorsal penile vein in in the sham operation group and the warm ischemi Inferior vena cava blood was taken at 6 hours, 1 day, 3 days and 5 days after operation to detect serum creatinine and urea nitrogen levels. Kidney tissue samples were taken after death. NF-kappa B and tumor necrosis factor-alpha (TNF-alpha) were detected by RT-QPCR. The expression of HMGB1 was detected by Western blot and the degree of renal injury was detected by immunohistochemistry.
Results: The levels of HGMB1 in warm ischemia-reperfusion group and EP group were higher than those in normal control group, and the levels of CRE and BUN in warm ischemia-reperfusion group and EP group were higher than those in EP group. The levels of B and TNF-alpha were significantly higher in warm ischemia-reperfusion group than in EP group, and the degree of renal injury in warm ischemia-reperfusion group and EP group.
CONCLUSION: The expression of HMGB1 was detected in all renal specimens. The expression of HMGB1 increased after warm ischemia-reperfusion injury, which resulted in the increase of CRE, BUN, TNF-a and NF-kappa B. EP could decrease the expression of HMGB1, CRE, BUN, TNF-a and NF-kappa B, and help to reduce the renal injury induced by warm ischemia-reperfusion.
The second chapter is about the expression and intervention of HMGB1 in cold ischemia reperfusion injury.
Objective: To explore the mechanism of cold ischemia-reperfusion injury induced by HMGB1 and to clarify the expression of HMGB1 in cold ischemia-reperfusion injury of kidney.
METHODS: Sixty healthy clean Sprague-Dawley (SD) male rats were randomly divided into sham-operation group, cold ischemia-reperfusion group and ethyl pyruvate (EP) treatment group. The right kidney cold ischemia-reperfusion injury model was established. After 6 hours, 1 day, 3 days and 5 days, the serum creatinine, urea nitrogen, NF-kappa B, tumor necrosis factor-alpha (TNF-alpha) and HMGB1 in renal tissue were detected by RT-QPCR and immunohistochemistry. Expression and severity of kidney injury.
Results: The levels of HGMB1 in cold ischemia-reperfusion group and EP group were higher than those in normal control group, and the levels of CRE and BUN in cold ischemia-reperfusion group and EP group were higher than those in EP group. The level of NF alpha was significantly higher than that of the normal control group, and the cold ischemia reperfusion group was higher than that of the EP treatment group.
CONCLUSION: The expression of HMGB1 was detected in all renal specimens. The changes of HMGB1, CRE, BUN, TNF-a and NF-kappa B in cold ischemia-reperfusion injury of kidney were the same as those in warm ischemia-reperfusion injury, but the increase degree was lower. Injury.
【学位授予单位】：中国人民解放军军医进修学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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