【作者】 袁芳；

【导师】 刘伏友；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病常见又严重的并发症之一,它是以肾小球系膜细胞增生,细胞外基质增多,肾小球基底膜增厚等病理生理改变及肾小球硬化为主要特征的病变。其发病机制复杂,与肾小球血流动力学改变、生化代谢紊乱,氧化应激,细胞因子、遗传易感性等多种因素有关。DN预后差,相比其它原因所致的肾脏疾病更难以治疗,其原因尚不清楚。研究表明,细胞因子分泌增多,细胞外基质(ECM)蛋白代谢异常在DN发病机制的各个环节起着重要作用,其中转化生长因子β1(TGF-β1)是一个重要的细胞因子,它的异常表达在ECM代谢过程中发挥重要的作用,可直接或间接促进ECM成分增加,是导致糖尿病患者肾脏纤维化的最后共同中介物质。此外,近来越来越多的证据表明,氧化应激(oxidative stress,OS)在糖尿病肾病发病中起重要作用；糖尿病状态下由于活性氧簇(reactive oxygen species,ROS)的产生增多和／或清除减少导致了氧化应激状态。ROS本身可攻击脂质、蛋白质和DNA,导致肾脏损害。脂联素(adiponectin,ADPN)是一种由白色脂肪组织分泌的蛋白产物,是脂肪组织在人血浆分泌最多的蛋白。脂联素又称30kD脂肪细胞补体相关蛋白(Acrp30)或28kD胶原结合蛋白(GBP28),人类脂联素基因由apM1 mRNA编码,位于染色体3q27上,全长16Kb,由3个外显子和2个内含子组成。自从1995年发现脂联素以来,多项研究已证实脂联素具有抗炎、改善胰岛素抵抗、抗动脉粥样硬化、降血糖、血脂等作用,最近研究发现脂联素还具有抗氧化作用。临床研究发现血浆ADPN浓度与糖尿病肾病关系密切。对早期糖尿病肾病的研究显示循环脂联素水平与尿蛋白呈负相关,说明早期糖尿病肾病内皮功能受损与低脂联素水平有关；进展期糖尿病肾病患者尿脂联素和血脂联素水平增加,认为其可能通过抗炎、抗动脉硬化等作用,减轻肾脏损害。然而脂联素对糖尿病肾病保护作用的机理研究甚少。因此我们在体外将脂联素作用于高糖诱导的人肾小球系膜细胞,观察其对细胞增殖、TGF-β1、活性氧簇(ROS)和内皮型一氧化氮合酶(eNOS)产生的影响,并对其相关信号通路进行探讨；同时构建表达ADPN的pIRES2-EGFP-gAd荧光质粒,将其转染糖尿病大鼠模型,探讨其对肾脏的保护作用与机理。本研究的目的、方法、结果、结论分三部分概述如下：第一章脂联素对系膜细胞氧化应激和TGFβ1的影响及细胞信号转导机制研究目的：观察脂联素对高糖诱导下肾小球系膜细胞ROS、eNOS以及TGF-β1产生的影响,并探讨腺苷酸活化蛋白激酶(AMPK)信号通路在这一过程中的作用。方法：体外培养人肾小球系膜细胞(HMCs),RT-PCR检测HMCs脂联素受体(adiponectin receptor,AdipoR)mRNA的表达。分别用高糖(30mM)和不同浓度(3-10ug/ml)重组人球形脂联素蛋白干预HMCs24、36小时, MTT试验观察细胞增殖情况。将细胞随机分为4组：对照组、高糖组(30 mM)、高糖+脂联素组和高糖+脂联素+AMPK抑制剂araA(adenine 9-β-D-arabinfuranoside)组,观察脂联素对高糖诱导的HMCs氧化应激和TGF-β1 mRNA和蛋白表达的影响。荧光探针检测ROS释放；RT-PCR检测eNOS和TGF-β1 mRNA表达,Western blot检测eNOS和TGF-β1蛋白水平。另外,为了进一步明确脂联素上述作用的细胞信号转导机制,用脂联素10ug/ml单独干预系膜细胞,分别于0、5、10、15、30min提取胞浆蛋白,Western blot检测磷酸化AMPK蛋白(p-AMPK)表达。结果：1)RT-PCR证实人肾小球系膜细胞上有AdipoR1和AdipoR2表达,半定量结果示AdipoR1明显占优势(AdipoR 1的表达强度是AdipoR 2的3.5倍)；2)脂联素呈时间和剂量依赖性地抑制高糖刺激下的HMCs增殖；3)Western blot检测p-AMPK蛋白证实脂联素可以时间依赖的方式诱导系膜细胞AMPK磷酸化；4)高糖组HMCs的ROS释放量较对照组明显增加,eNOS产生减少,TGF-β1表达上调(p<0.05)；脂联素处理后ROS产生量较高糖组明显减少,eNOS表达上调,TGF-β1产生减少(p<0.05)；加入AMPK抑制剂araA后,可部分抑制脂联素的上述作用。结论：1、脂联素能够抑制高糖诱导的HMCs增生和活性氧产生,促进eNOS产生,下调TGF-β1的表达,从而对抗高糖对系膜细胞的损伤。2、脂联素可刺激系膜细胞AMPK通道开放,脂联素对HMCs的保护作用部分是通过脂联素受体介导AMPK信号通路实现的。第二章表达ADPN的pIRES2-EGFP-gAd质粒载体的构建及其在大鼠肾脏的表达目的：构建表达脂联素的pIRES2-EGFP-gAd质粒,观察其在大鼠肾脏的表达,为体内观察脂联素对糖尿病大鼠模型的肾脏影响奠定基础。方法：根据GENBANK中人脂联素的cDNA编码球形结构域(gAd)的序列设计引物,以已有的pET/gAd质粒(编码脂联素球形结构域的细菌表达质粒,gAd基因克隆于pET-15b载体的多克隆位点,长440bp)为模板,PCR扩增目的片段,连接T载体后转化大肠杆菌,用EcoRI和BamHI双酶切后,与pIRES2-EGFP荧光质粒连接,重组构建pIRES2-EGFP-gAd质粒。将构建成功的pIRES2-EGFP-gAd质粒采用脂质体介导经腹腔注射正常大鼠体内,然后在不同时间点(24h、48h、96h、7d)留取肾脏标本进行冰冻切片,在荧光显微镜下观察绿色荧光蛋白在肾组织中的表达情况,同时采用Western Blot检测肾皮质绿色荧光蛋白的表达。结果：1)重组构建的pIRES2-EGFP-gAd载体经双酶切电泳分析及DNA测序证实无碱基突变。2)在腹腔注射脂质体/pIRES2-EGFP-gAd转染复合物后24h,荧光显微镜下观察,即可在肾小球肾间质检测到绿色荧光蛋白的表达,在注射后48h荧光强度进一步增强,随着给药时间的延长,绿色荧光蛋白在肾脏的表达逐渐减少,在注射后第7d时,虽然仍能检测到绿色荧光蛋白的表达,但荧光强度明显减弱。Western Blot检测EGFP蛋白结果与冰冻组织切片结果一致。结论：脂质体能够介导pIRES2-EGFP-gAd真核载体在大鼠肾脏表达,实验中将能发出绿色荧光的EGFP报告基因融合在gAd基因的3’端,既保留了gAd的生物活性,又便于基因治疗中可检测到蛋白表达,为探讨脂联素在DN中的作用提供实验模型奠定基础。第三章复制DM模型研究脂联素对肾脏的保护作用及机制目的：研究脂联素对糖尿病大鼠的肾脏保护作用及其相关机制。方法：采用高糖高脂饮食配合腹腔注射链脲佐菌素(STZ)的方法复制糖尿病大鼠模型。将32只Wistar大鼠随机分为正常对照组(NC组)、糖尿病组(DM组)、ADPN干扰组(DA组)和空载体转染组(DP),其中DA, DP组分别腹腔注射脂质体/pIRES2-EGFP-gAd或脂质体／pIRES2-EGFP复合物。处理8周后,观察血糖、糖化血红蛋白(HbA1c)、24h尿微量白蛋白(UMA)变化；留取肾组织切片观察光镜下的病理改变并测定肾组织ROS释放量的变化；荧光定量RT-PCR检测各组大鼠肾组织eNOS、TGF-β1的基因转录水平,采用免疫组化法检测eNOS、TGF-β1、p-AMPK蛋白的表达水平。结果：与正常对照组比较,DM模型组大鼠UMA显著上升,肾组织ROS释放量增加(P<0.05),DM、DA、DP各组之间UMA无明显差异(P>0.05)。与DM组相比,DA组血糖和HbAlc有所下降,ROS释放明显减少(P<0.05)。光镜下DM组大鼠大部分肾小球系膜区增宽,基质大量增生,节段性基底膜增厚、部分肾小管上皮细胞空泡变性、脱落,肾小球内细胞数显著增多、单个核细胞浸润明显,与DM组相比,脂联素干预组以上病变均有减轻。与对照组相比DM组肾组织eNOS水平下降,TGF-β1表达明显上调,p-AMPK蛋白表达下调(p<0.05)；脂联素干预组eNOS水平也有下降,但较DM组升高,TGF-β1水平较DM组下降(P<0.05),p-AMPK蛋白表达明显高于DM组(P<0.05)。结论：脂联素对糖尿病大鼠肾脏具有保护作用,其机制一方面通过刺激AMPK磷酸化,抑制ROS产生,减轻氧化应激反应,上调糖尿病大鼠肾组织中eNOS的表达,另一方面通过下调TGF-β1基因,减轻ECM在肾间质的堆积而实现的。更多还原

【Abstract】 Diabetic nephropathy(DN) is one of the major complications of diabetes mellitus, it’s characteristic with mesangial expansion、extracellular matrix deposition、basement membrane thickening of glomeruli and glomerulosclerosis.The pathogenesis of DN is unkown,it associates with altered glomerular hemodynamics、metabolic disorder、oxidative stress、cytokines and hereditary susceptibility.Diabetic nephropathy has worse outcomes and is more difficult to be treated than nephropathy caused by other diseases, the reasons are unclear.Resent studies have shown that the increased secretion of cytokines and over expression of extracellular matrix(ECM) protein play the important roles in the progression of diabetic nephropathy.Among them, transforming growth factor (31 (TGF-β1)is an important cytokine, which directly or indirectly promote the components of ECM, leading to renal fibrosis in patients with diabetes.Resently,more evidences show that oxidative stress (OS) plays an key role in the development of diabetic nephropathy. Overproduction and less consumption of reactive oxygen species(ROS) resulting in oxidative stress,ROS itself can attack lipids, proteins and DNA, leading to kidney damage.However the mechanisms by which ROS production induce kidney damage need to be addressed.Human adiponectin(ADPN) is a novel adipose-derived adipocytokines.In humans,adiponectin is the most abundant gene transcript proteins in adipose cells. It is also known as complement-related protein (Acrp30) or 28KD collagen binding protein (GBP28).The human adiponectin gene is encoded by apM1 mRNA, located on 3q27,a total length of 16Kb.It is composed by three exons and two introns. ADPN has been shown to have anti-atherosclerosis and anti-inflammatory properties as well as regulation of insulin-sensitizing metabolic effects and vascular protective properties.In 1995,Scherer et al first cloned and identified ADPN.Recently, many researchers confirmed that anti-oxidation properties also have been attributed to adiponectin. On the other hand,clinical studies have demonstrated that plasma concentrations of ADPN are closely related with progression of diabetic nephropathy. Furthermore,circulating adiponectin levels were negatively correlated with proteinuria in type-2 diabetics in early stage,which shows that endothelial dysfunction is associated with low circulating adiponectin. The urinary and serum adiponectin increased in patients with advanced diabetic nephropathy,these data suggest that ADPN may be through regulation of anti-inflammatory,、anti-atherosclerosis to reduce kidney damage and delay the progression of diabetic nephropathy.However,little is known about the protective mechanism of adiponectin on diabetic nephropathy.Therefore, in the present study, we observed the effects and mechanism of recombinant globular adiponectin (gAd) on the generation of TGF-β1、ROS and endothelial NO Synthase (eNOS) in vivo and in vitro.The specific aims are addressed in there thesis.The objective、methods、result and conclusion are listed here. Chapter one Studies of the effects of Adiponectin on oxidative stress and the expression of TGFβ1 in HMCs and involved Cell Signal PathwayObjective:To investigate the effects of adiponectin on high glucose induced ROS、eNOS and TGF-β1 expression in human mesangial cells(HMCs), and study the role of Adenosine Monophosphate Kinase (AMPK) signal pathway in adiponectin actions.Methodes:AdipoR mRNA expression were detected in cultured HMCs by RT-PCR. Cell proliferation was assessed using the MTT assay method.HMCs were incubated with 30mM high glucose and different concentrations of globular adiponectin (gAd,3-10ug/ml)for 24h and 36h to investigate the dose dependent effect.Then the cells were randomly assigned into four groups:the control group, the high glucose group(30mM,HG),HG+adiponectin group, HG+adiponectin+AMPK inhibitor(araA) group.The generation of ROS was detected by fluorescence probe, eNOS and TGF-β1 expression were determined using RT-PCR and Western blot respectively.Furthermore,to define the involved signal pathway we incubated the HMCs with gAd for 0-30minute to detect the expression of AMPK and phosphorylated AMPK by Western blot.Result:1)The expression of AdipoR were exhibitted in HMCs,and the AdipoR2 expression was more than that of AdipoRl for 3.5fold; 2)Adiponectin significantly inhibited cell proliferation in a dose-and time-dependent manner;3)Treatment of HMCs with adiponectin resulted in a time-dependent increased phosphorylation of AMPK;4) Compared to HG group, Cells treated with gAd showed high glucose-induced ROS release inhibited, the expression of eNOS up-regulated and the expression of TGF-β1 decreased (p<0.05).The effects of gAd were partly blocked by AMPK inhibitor araA.Conclusions:Adiponectin inhibits cell proliferation and inhibits generation of ROS induced by high glucose in HMCs.Furthermore, adiponectin also stimulates eNOS activity and down regulates the expression of TGF-β1.The mechanism is partly through activation of AMPK signal passway mediated by AdipoRl. Chapter two Construction of eukaryotic expression plasmid pIRES2-EGFP-gAd and analysis its expression in rat kidneyObjective:To construct the eukaryotic expression plasmid pIRES2-EGFP-gAd, and observe the expression of gAd in rat kidney.Methodes:The gAd cDNA fragments were obtained by PCR from pET/gAd(Plasmid which contains a full length cDNA of gAd).Then, the PCR product was linked with T-vector and translated into JM109.It was digested by two restrictive endonuclease,then the gAd cDNA was collected and recombined with eukaryotic expression vector pIRES2-EGFP by using gene recombination technique.The recombined plasmid pIRES2-EGFP-gAd was transfected into normal rat kidney with Lipofectamine Transfection Reagent by intraperitoneal injection. The kidney tissues were collected at different time points (24h,48h,96h,7d) after injection,gAd/GFP green fluorescence protein expression was determined by fluorescence microscopy and Western Blot respectively.Result:1)The pIRES2-EGFP-gAd expression plasmid was constructed successfully.2) The gAd/GFP green fluorescent protein was detected at the glomeruli and tubular in the 24th hour after injection and the fluorescence intensity became stronger in the 48th hour. The level of fluorescence protein expression became gradually weakened in the 7th day. In Western Blot test, same results were observed. Conclusions:The pIRES2-EGFP-gAd gene was expressed in rat kidney successfully by Lipofectamine Reagent with intraperitoneal injection.In the experiments we fused a green fluorescent reporter gene of the EGFP gene in the 3’end of gAd, not only retained the biological activity of gAd but also could easy to detect protein expression in gene therapy.These provide experimental basis for further investigating of adiponectin on diabetic nephropathy. Chapter three In vivo studies the role of adiponectin on the renal protective effect in diabetic nephropathyObjective:To investigate the reno-protective effect of adiponectin on renal damage in streptozotocin(STz)-induced diabetic rats and its mechanisms.Methodes:Diabetes mellitus models were induced by high-lipids and high-sucrose feeding plus STZ intraperitoneal injection. The recombinant plasmid of pIRES2-EGFP-gAd was induced into rat models mediated by transfection reagent with intraperitoneal injection.32 wistar rats were randomly assigned into four groups:the normal control group(NC);diabetes group without any therapy(DM);the diabetes group treated with pIRES2-EGFP-gAd(DA) and the diabetes group treated with pIRES2-EGFP(DP).After corresponding treatments for 8 weeks,blood glucose、HbA1c and urinary microalbumin(UMA) were measured.The kidneys were collected to test the generation of reactive oxygen species (ROS).The renal pathologic changes were observed by light microcopy.The mRNA expression of eNOS and TGF-β1 were determined by real-time PCR;the protein of eNOS、TGF-β1 and p-AMPK were assessed by immunohistochemical method.Result:UAER and generation of ROS were increased in DM group as compared with control group(P<0.05),while there was no significant differences in UARE among the DM、DA、DP groups(P>0.05).Blood glucose level、HbA1c and generation of ROS were decreased in DA group as compared with group DM(P<0.05).Compared with control group,there were glomerular hypetrophy, mesangial expansion, basement thickening,tubular epithelial cells cavitation and exfoliation, and mononuclear lymphocyte infiltration in DM group.While these changes were ameliorated in gAd transfection group. The expression of eNOS and p-AMPK in DM group were decreased, the expression of TGF-β1 was increased compared with control group(p<0.05),but in gAd transfection group the expression of eNOS and p-AMPK increased compared with DM group(p<0.05),and the expression of TGF-β1 was even lower than DM group(p<0.05).Conclusions:Adiponectin has reno-protective effect on diabetic rat kidney.The mechanisms of it are correlated with stimulating the AMPK signal passway, inhibiting the generation of ROS,relieving oxidative stress,up-regulating the expression of eNOS in renal tissues of diabetic rats;on the other hand,it also can down-regulate the expression of TGF-β1,reduce the accumulation of ECM in the renal interstitial.更多还原