【作者】 王蕾；

【导师】 关广聚；

【作者基本信息】 山东大学 ， 内科学， 2012， 博士

【摘要】 研究背景和目的糖尿病肾病(diabetic nephropathy, DN)是糖尿病最常见的慢性并发症之一,也是终末期肾脏病(end stage renal disease, ESRD)患者死亡的主要原因。目前糖尿病肾病的治疗原则主要是控制血糖、血压,控制饮食和改善肾功能,但临床上糖尿病肾病患者一旦进入临床蛋白尿期,尽管基于各种手段,DN患者的肾功能还是会逐渐减退,逐渐发展至ESRD,由此带来的透析、肾移植等治疗费用在整个医疗支出中所占比例正逐年增长,给病人和社会造成极大的经济负担。目前关于DN发病机制的研究既往多集中在血糖、血脂代谢紊乱与血流动力学异常等方面。近年研究发现炎症反应参与DN的发生和发展。肾小球系膜细胞(MCs)和系膜基质增多是糖尿病肾小球硬化的主要病变,MCs分泌细胞因子和细胞外基质,参与肾小球炎症反应过程,促进肾小球硬化,因此研究药物对炎症状态下肾小球MCs增殖和分泌,在糖尿病肾病的治疗中具有十分重要的意义。促红细胞生成素(Erythropoietin, EPO)主要是由成人肾脏产生的一种糖蛋白激素,能特异性地作用于哺乳动物的红系祖细胞,促进其增殖、分化并抑制其凋亡。自人工合成以来一直用于治疗各种疾病导致的贫血,特别是肾性贫血的治疗。EPO通过与EPO受体(EPOR)结合启动细胞内信号转导通路起作用。近年来,EPO的细胞保护作用和器官保护作用备受关注,EPOR不仅存在于红系生成细胞,还存在于全身各组织器官。重组人红细胞生成素(rHuEPO)被认为是一种全身性的、心管系统和肢体保护性细胞因子。其中rHuEPO对神经系统和心血管系统研究较多,临床上已用于神经系统、心血管系统和肢体缺血性疾病的治疗,在肾脏保护方面,目前对急性肾损伤的保护作用机制研究较为透彻的有抗氧化应激、抗凋亡、抗炎症反应。关于EPO在糖尿病肾病的保护作用,研究较少,最近国内外有文献报道EPO对糖尿病肾病动物模型的保护作用。关于EPO对高糖培养的大鼠肾小球系膜细胞的作用目前未见有报道,肾脏是EPO的主要产生部位,且EPO及受体在肾缺血再灌注损伤及毒性物质损伤时表达上调,文献证实EPO受体存在肾小球系膜细胞、内皮细胞和肾小管上皮细胞,表明EPO具有内源性肾脏保护作用。本实验通过体外系膜细胞培养技术,给予高糖环境下,rHuEPO刺激,用流式细胞技术、实时荧光定量PCR及Western bolt等分子生物学技术,观察系膜细胞的形态、细胞增殖和凋亡,检测细胞凋亡相关基因Bcl-2(B-Cell Lymphoma)、caspase-3蛋白的表达,探讨rHuEPO对高糖培养的肾小球系膜细胞增殖及凋亡的影响并分析可能的机制。材料与方法1、体外培养肾小球系膜细胞,分别培养于正常和高糖葡萄糖浓度环境中,采用CCK-8比色法观察24、48、72小时MCs增殖的情况。选择高糖组浓度为30mmol/L,系膜细胞分组为：空白对照组(NC)、高糖组(HG),高糖和不同浓度的rHuEPO (1、10、50、100、1000IU/ml)干预组,作用24、48、72小时。CCK-8比色法检测细胞增殖能力,观察不同浓度、不同时间rHuEPO对高糖培养的系膜细胞增殖的影响；倒置显微镜动态观察系膜细胞形态和增殖的变化。2、应用ELISA法和实时荧光定量PCR方法检测细胞单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)、转化生长因子-β1(transforming growth factor-β1,TGF-β1)蛋白和mRNA的表达。3、应用流式细胞仪(FCM)检测rHuEPO对高糖培养的系膜细胞凋亡的影响。4、Western bolt检测caspase-3和Bcl-2蛋白的表达情况。结果1、不同浓度葡萄糖、rHuEPO对系膜细胞增殖的影响在30mmol/L以下时,不同浓度的葡萄糖培养系膜细胞24、48、72小时发现,葡萄糖对系膜细胞的增殖呈浓度和时间依赖性,以30mmol/L培养72小时反应最佳,40mmol/L和50mmol/L的葡萄糖有抑制系膜增殖的效应。选择高糖组葡萄糖浓度为30mmol/L,分别加入不同的rHuEPO浓度,rHuEPO在低于100IU/ml能促进高糖诱导的MCs增生,呈剂量和时间依赖性,rHuEPO在1000IU/ml时,细胞毒性明显,大部分细胞死亡2. ELISA法检测细胞培养上清MCP-1、TGF-β1含量,real time FQ-PCR方法检测细胞MCP-1、TGF-β1mRNA的表达正常培养的系膜细胞MCP-1、TGF-βmRNA有低水平的表达,高糖组刺激24、48、72小时均有高水平的表达,rHuEPO在1、10、50、100IU/ml时,作用于高糖培养的MCs,细胞MCP-1、TGF-β1mRNA的表达均显著降低。3、各组系膜细胞的凋亡情况对照组系膜细胞有一定的凋亡率,高糖组系膜细胞早期凋亡和晚期凋亡明显增加,rHuEPO在1、10、50、100IU/ml时,作用于高糖培养的MCs,可抑制高糖培养系膜细胞的凋亡。4、系膜细胞Bcl-2和Caspase-3的表达情况正常对照组系膜细胞表达高水平的Bcl-2和低水平的Caspase-3,高糖组作用后Bcl-2表达降低,Caspase-3表达升高,较对照组差异有统计学意义,rHuEPO在1、10、50、100IU/ml时,Bcl-2蛋白表达量增加,Caspase-3蛋白表达量降低,与高糖组比较有统计学意义。结论1、高糖在一定浓度和时间内有刺激系膜细胞增殖的效应,大剂量1000IU/ml rHuEPO对高糖培养的系膜细胞有细胞毒性,小剂量(1-100IU/m1)rHuEPO可刺激高糖培养的系膜细胞的增殖,呈浓度和时间依赖性。2、高糖增加系膜细胞MCP-1、TGF-β1蛋白和mRNA的表达,1-100IU/ml rHuEPO能抑制高糖培养的MCs MCP-1、TGF-β1蛋白和mRNA的表达。3、高糖培养的系膜细胞凋亡增加,rHuEPO能抑制高糖培养的MCs的凋亡,可能与凋亡相关的基因Bcl-2表达上调,Caspase-3表达下调有关。研究背景和目的贫血是终末期肾脏病(ESRD)血液透析(HD)患者最常见的并发症,贫血可以引起患者组织氧供减少、左室肥厚(LVH)、充血性心力衰竭(CHF)、肌肉易疲劳、运动耐力和机体免疫力下降等,ESRD HD患者死亡率升高。重组人促红细胞生成素(rHuEPO)在临床的广泛应用大大减少了肾性贫血的发生率,大部分肾性贫血得到了有效的治疗,但rHuEPO的反应性在不同的患者表现不同,大约90-95%的患者对rHuEPO有良好的反应性,血红蛋白水平能维持在11-12g/dl或以上水平,但5-10%的ESRD患者rHuEPO反应性差或无反应,血红蛋白水平不能维持在11-12g/dl或以上水平,存在明显的rHuEPO抵抗。文献报道,许多因素参与rHuEPO抵抗的发生,包括铁缺乏、慢性炎症、急性感染、失血、甲状旁腺功能亢进、VitB12和叶酸缺乏等。其中最常见的一个原因是铁缺乏。其中包括了铁的绝对不足以及功能性铁缺乏。功能性铁缺乏是指体内铁储备充分,而铁不能被充分利用。但一部分患者铁补足后,仍存在rHuEPO抵抗。rHuEPO抵抗的另一个重要原因归因于慢性炎症,但这些因素排除后,仍有一些患者存在rHuEPO抵抗。Hepcidin是一种由肝脏细胞产生的由25个氨基酸组成的具有广谱杀菌抑菌作用的抗菌肽,属于防御性蛋白家族。近年研究发现铁调素(Hepcidin)在维持铁代谢平衡中起着关键性的调控作用,参与多种铁代谢疾病的发病机制。Hepcidin可以在患者尿液中和超滤液中检测到。文献报道体内铁负荷、慢性炎症和IL-6可以使hepcidin水平升高,贫血,缺氧和rHuEPO治疗可以使hepcidin水平下降。Hepcidin与膜铁转运蛋白(FP1)结合发挥作用,后者在十二指肠、肝细胞及巨噬细胞存在高表达,FP1水平降低时,hepcidin可抑制铁的释放,hepcidin在慢性病的贫血中发挥重要作用。近年研究发现细胞因子介导的炎症抑制骨髓红细胞的生成为ESRD患者贫血的一个重要原因,表现为C反应蛋白(CRP)升高,认为慢性炎症可能为rHuEPO抵抗性贫血的一个重要原因。HD透析患者慢性炎症可以调节hepcidin生成,血液透析患者hepcidin水平升高。但是hepcidin在肾性贫血中的发病机制仍不是很清楚。有研究表明ESRD患者hepcidin在体内蓄积,HD患者不同的透析方式可对hepcidin的水平产生影响。目前由于hepcidin分子量小,检测较困难,对设备要求较高,限制了广泛应用,我们通过测定其前体pro-hepcidin水平了解体内hepcidin水平。该课题通过临床观血液透析肾性贫血患者与促红素抵抗患者体内pro-hepcidin的水平、炎症因子(hs-CRP)以及铁代谢状态,研究pro-hepcidin与rHuEPO抵抗肾性贫血、慢性炎症及铁代谢状态的相互关系,观察pro-hepcidin是否参与rHuEPO抵抗肾性贫血的发病,为将来接受EPO治疗患者提供有效的诊断和决策参考。研究目的如下：1、ELISA法测定MHD患者、rHuEPO抵抗性贫血患者及健康对照者血清pro-hepcidin水平。2、分析ESRD MHD患者rHuEPO抵抗与铁代谢、慢性炎症和pro-hepcidin之间的关系。3、ROC曲线预测hepcidin对rHuEPO抵抗的诊断价值。材料与方法1.MHD患者40例入选,其中20例为rHuEPO反应良好组,20例为rHuEPO抵抗组,另选择20例健康体检者作为对照组。2.一般资料采集：血液透析患者均于透析前动静脉瘘处取血，健康对照组于清晨空腹取肘静脉血。分别检测血红细胞计数，血红蛋白浓度，红细胞积压，及肝肾功能等生化指标，pro-hepcidin、hs-CRP、铁代谢指标。3.测定指标：HD患者和健康对照常规检测血液学指标及生化指标,铁代谢指标包括血清铁蛋白、血清铁和总铁结合力(计算转铁蛋白饱和度)、iPTH、叶酸和VitB12, MHD患者测定透析前后血尿素氮和肌酐水平。4.检测方法：(1)血清Pro—hepcidin:ELISA方法(2)血清hs-CRP:透射比浊法测定。(3)血清铁蛋白：微粒子发光法检测。(4)血清铁和总铁结合力：亚嗪比色法测定,计算出转铁蛋白饱和度(TSAT) TSAT=(血清铁/总铁结合力)×100。(5)叶酸和VitB12:微粒子酶联免疫法检测。(6)iPTH：电发光法检测。(7)透析充分性计算：Kt/V=-ln(R-0.008t)+(4-3.5R)UF/W URR=100(1-Ct/Co)(8)常规检测血脂、肾功、肝功等生化指标、血细胞各项参数。5.统计方法：应用SPSS13.0软件包进行统计学处理,符合正态分布或近似正态分布的计量资料以x±s表示,用独立样本t检验分析组间差别,计数资料采用χ2检验,影响EPO反应性的因素采用Spearman等级相关分析,以P<0.05表示差异有统计学意义,多元逐步回归分析影响pro-hepcidin的因素,ROC曲线预测pro-hepcidin对EPO抵抗的诊断价值。结果HD患者血清铁蛋白、血清pro-hepcidn、hs-CRP明显高于正常患者,TF明显低于正常患者,EPO抵抗的患者血清铁蛋白、血清pro-hepcidin、hs-CRP明显高于反应正常的患者。血清pro-hepcidin水平与血清铁蛋白和hs-CRP密切相关,多元逐步回归分析显示影响血清pro-hepcidin的因素有SF、SI和hs-CRP,预测EPO抵抗诊断价值的ROC曲线示pro-hepcidin、SF和hs-CRP的曲线下面积分别为0.713、0.769、0.958,其中hs-CRP预测价值最大。结论HD患者pro-hepcidin水平明显升高,rHuEPO抵抗与慢性炎症、铁代谢水平密切相关,血清pro-hepcidin、SF、hs-CRP是rHuEPO抵抗的标志。更多还原

【Abstract】 Background and objectiveDiabetic nephropathy(DN) is one of the most common chronic complications of diabetes mellitus and the major cause of end-stage renal disease(ESRD). It is widely accepted for the time being that therapeutic principles for DN are as follows:the control of hyperglycemia, blood pressure and diet as well as renal function protection. Renal function would decline quickly and progress into ESRD when clinical proteinuria appears in diabetic patients. Thus the expenditure on dialysis and kidney transplantation increases every year and causes heavy economical burdens on the patients and society. It is considered traditionally that an interaction of metabolic and hemodynamic factors involved in the development of DN. However, the recent study has demonstrated that inflammation is an important cause in chronic progressive renal disease in diabetic patients. Mesangial cell (MCs) proliferation and extracellular matrix accumulation are the main causes of diabetic nephropathy. MCs secrete cytokines and extracellular matrix, and induces glomerulosclerosis. So studying MCs proliferation and secretion is important in the therapy of proliferating glomerular disease.Erythropoietin is a glycoprotein hormone produced in adults primarily in the kidneys. It was originally understood to be an erythropoietic stimulator, regulating the differentiation and maturation of erythroid progenitor cells and preventing apoptosis of developing erythroid progenitor cells. The recombinant forms of the hormone have been used to effectively treat anemia in many diseases such as chronic inflammation, cancer especially renal insufficiency. The hormone’s effects are signaled through the erythropoietin receptor and increasingly understood transduction intermediates. Recently studies have suggested that erythropoietin could act as tissue cell and organ protective molecule. The erythropoietin receptor has been found not only on hematopoietic tissues but also on many none-hematopoietic tissues. It is well established that EPOR has been found in kidney tissue, including mesangial and renal tubular cells and endothelial cell. Many studies confirmed that EPO plays a significant renoprotective when administered to animal models with acute renal injury, ischemia reperfusion injury and cisplatin-induced kidney damage. Further, some studies showed that EPO may have protective effects for chronic kidney disease such as DN, but whether EPO can affect MCs under high glucose is still unclear.In the present study, by cck-8, real time RT-PCR, Western blot and FCM, we investigated the potential effect of recombinant human erythropoietin on high glucose induced renal mesangial cell damage, the morphological change of renal mesangial cell, inflammation marks, apoptosis index and expression of Bcl-2and Caspase-3protein expression.Methods1. Rat MCs were cultured with the medium which contains of normal glucose (NC), different concentrations of high glucose and30mmol/L high glucose (HG) with1,10,50,100,1000IU/ml of rHuEPO (E1-E5). Cell proliferation was tested with CCK-8and observed by spectrometry24,48and72h later.2. The concentration of MCP-1and TGF-β1in supernatant were measured by ELISA. The mRNA expressions of MCP-1and TGF-β1in cells were determined by real-time RT-PCR.3. MCs apoptosis index were measured by flow cytometry with fluorescene-activated cell sorter analysis. 4. Apoptosis associated gene Bcl-2and Caspase-3expressions in MCs were detected by Western blot.Results1. Glucose and rHuEPO on proliferation of MCsThe MCs proliferation presented a time-and dose-dependent feature under30mmol/L glucose, and got to the highest point at the concentration of30mmol/L for72hours.40mmol/L and50mmol/L glucose could inhibit the proliferation of MCs. RHuEPO (I,10,50,100IU/ml) could inhibit the high glucose induced proliferation in a time-and dose-dependent feature. RHuEPO of1000IU/ml could have cytotoxicity on high glucose induced MCs.2. MCP-1、TGF-β1protein and mRNA expression in MCsThere had low level MCP-1and TGF-β1protein and mRNA expression in normal MCs. High glucose could stimulate the synthesis of MCP-1and TGF-β1protein and mRNA. Different concentrations from1-100IU/ml of rHuEPO could inhibit MCP-1and TGF-β1protein and mRNA expression significantly.3. Apoptosis in MCsThe percentage of apoptotic MCs cell stimulated by30mmol/L glucose significantly increased compared with control. When the cells were incubated with30mmol/L glucose and rHuEPO (1,10,50,100IU/ml) for24,48,72h, the percentage of apoptotic MCs cell decreased significantly.4. High glucose (30mmol/L) significantly decreased Bcl-2mRNA and protein expression and increased Caspase-3protein.The presence of rHuEPO, dose (1,10,50,100IU/ml) dependently prevented high glucose induced decreases in Bcl-2and increases in Caspase-3.Conclusion1. High concentrations of glucose can stimulate MCs proliferation with a time-and dose-dependent feature. Large dose of rHuEPO (1000IU/ml) has cytotoxic effect on high glucose induced MCs.(1-100IU/ml) rHuEPO can promote high glucose induced proliferation of MCs also in time-and dose-dependent way. 2. High glucose contributes to the upregulation of MCP-1and TGF-βprotein and mRNA in rat MCs.1-100IU/ml rHuEPO can inhibit expressions of MCP-1and TGF-Pprotein and mRNA in high glucose induced MCs. These findings provide experimental evidence for further study of the possibly protective effect of rHuEPO against DN.3. High glucose can promote apoptosis in MCs. RHuEPO can suppress apoptosis of high glucose induced MCs. The effects of rHuEPO on MC apoptosis perhaps correlate with upregulation of Bcl-2and downregulation of Caspase-3. BackgroundAnemia is among the most frequent complications associated with HD. Anemia can lead to a reduction in tissue oxygenation, increase in cardiac output, left ventricular hypertrophy, congestive heart disease, fatigue, reduction in exercise capacity and immunodeficiency, and eventually a higher mortality. The incidence of renal anemia has reduced significiantly in maintenance hemodialysis (MHD) patients due to rHuEPO use. Many patients can be treated successfully with recombinant human erythropoietin (rHuEPO). However, the responses to EPO varies among individual patients. According to the literature,90-95%of renal anemia responded in a dose-dependent manner to EPO, whereas the remaining5-10%of patients had a blunted or no response to EPO, despite high-dose therapy. These patients are rHuEPO-resistant. Various factors have been reported to be related to this hypo-responsiveness including iron deficiency, inflammation, infection, blood loss, hyperparathyroidism, aluminum toxicity, and vitamin B12and folate deficiencis. The main cause of resistance is iron deficiency and inflammation status, but rHuEPO-resistant anemia persists in some HD patients after exclusion of these factors.A recent study showed that a newly discovered mediator of innate immunity, hepcidin, is a key regulator of iron homeostasis. Hepcidin is a defensin-like peptide of25amino acids that is produced mainly in liver cells and can be detected in human urine and ultrafiltrates. Hepcidin production is enhanced by iron overload, inflammation and IL-6, and suppressed by anemia, hypoxemia and rHuEPO treatment. Hepcidin binds to ferroportin, a cellular iron exporter that is highly expressed in duodenal and liver cells and macrophages. Therefore, hepcidin suppresses extracellular release of iron by decreasing ferroportin. It has been proposed that hepcidin is a primary factor in the pathogenesis of anemia of chronic diseases (ACD).It has been reported that cytokine-induced inflammation suppressed bone marrow erythropoiesis in HD patients and is a possible cause of anemia. Panichi et al. observed a higher level of CRP in HD patients. It is therefore suggested that HD patients have a chronic inflammatory status and ACD may be one of the causes of rHuEPO-resistant anemia in these patients. Hepcidin synthesis is regulated by inflammation in HD patients, being enhance in non-responders compared with responders. Moreover, increased levels of prohepcidin have been reported in HD patients.However, the relationship between rHuEPO-resistant anemia, inflammation and hepcidin in HD patients has not been studied. In the present study, we investigated whether prohepcidin is involved in rHuEPO-resistant anemia in HD patients.Objective1. To determine serum pro-hepcidin in rHuEPO resistant anemia, ESRJD patients and healthy controls.2. To analyze the relationship between rHuEPO resistant anemia,inflammation markers and pro-hepcidin.3. To predict the value of pro-hepcidin in rHuEPO resistant anemia by ROC curve.Methods1. Forty HD patients and twenty healthy controls were enrolled in the study. Among HD patients,20were hyporesponsive to rHuEPO therapy and20were normal responsive to rHuEPO therapy.2. Blood sample collection was performed in all HD patients immediately before the start of HD. Blood sample collection was performed in healthy controls on an empty stomach.3. Complete blood red cell count, Hb concentration, hematocrit and serum ferritin (SF), Serum iron (SI), total ironbinding capacity (TIBC), saturation rate of transferrin (TSAT), transferrin (TF), hyper-sensitive C-reactive protein(hs-CRP), pro-hepcidin were measured in all patients and controls.Hematometry including Red blood cell(RBC), hematocrit, Hb concentration, hematological indices were performed with a Sysmex2100automatic analyzer (Japan) and biochemical analyses were performed with a Hitachi7060automatic analyzer (Tokyo, Japan), Post-dialysis blood urea nitrogen (BUN) levels were measured to calculate the delivered dialysis dose (Kt/V) using the Daugirdas method. TSAT was calculated as [(serum iron/TIBC)×100].Plasma concentrations of pro-hepcidin were determined using an enzyme-linked immunosorbent assay. The plasma concentrations of high sensitive CRP (hs-CRP) was determined using turbidimetry, according to the procedure recommended by the manufacturer. Serum pro-hepcidin and hs-CRP were measured using a Pro-hepcidin ELISA kit (Competition ELISA; DRG International Inc, Germany) and Bekman automatic chemical analyzer respectively. Serum ferritin, transferrin concentration and total iron binding capacity were determined using Bekmen AccEss automatic immune analyzer according to the procedure recommended by the manufacturer. Serum iron concentration was mearsured using colorimetric method.ResultsSerum levels of SF, pro-hepcidin and hs-CRP were higher in HD patients than in controls. While Serum TF was lower in HD patients than in controls. Serum levels of SF, pro-hepcidin and hs-CRP were high in rHuEPO resistant patient. Serum pro-hepcidin levels correlated positively with SF and hs-CRP in HD patients and HD1patients.ConclusionA close interaction exists between inflammation, iron status and prohepcidin serum levels that ultimately regulate intracellular iron availability. Prohepcidin and SF, together with hs-CRP, may prove to be good markers of resistance to rHuEPO therapy in HD patients.更多还原