【作者】 周桂凤；

【导师】 蒋云生；

【作者基本信息】 中南大学 ， 肾脏病内科， 2008， 博士

【摘要】 由于环境铅污染的普遍性和肾脏对铅毒作用的敏感性,目前铅性肾病的危害在国际、国内都是较普遍的。铅对肾脏的毒性作用是一个隐匿而渐进的过程,早期是亚临床型的,可逆的,发展到一定阶段即不可逆转。研究资料显示铅对肾脏的毒作用部位主要在肾近曲小管。铅性肾病病理学改变早期主要在肾小管上皮细胞内形成包涵体、损伤肾小管上皮细胞,继而逐步出现肾小管萎缩、肾单位丢失、肾间质纤维化导致肾功能低下。但目前对铅的肾脏主要靶细胞.肾小管上皮细胞损伤的细胞机制、分子机制知之甚少。本课题首先以正常人肾小管上皮细胞系（HK-2）为研究对象,观察醋酸铅致HK-2细胞凋亡、转分化作用,其剂量反应关系,以及对细胞凋亡、转分化信号通路相关分子的影响和碘化钾的干预作用。在此基础上,本课题运用功能蛋白质组学方法,分离鉴定与铅有高亲和性的结合蛋白,探讨铅致HK-2细胞凋亡、转分化的内在原因。以往的研究显示,铅可在细胞内与蛋白质形成高亲和性的铅结合蛋白（PbBPs）,PbBPs的形成与铅在肾脏引起的一种特征性病理改变-肾小管上皮细胞质、细胞核内形成包涵体有关。研究提示,铅穿过细胞膜,在细胞内沉积、迁移以及发挥其毒作用,是以铅结合蛋白结合的形式来进行的。所谓铅结合蛋白是指与铅有高亲和结合能力,与细胞对铅的适应性、反应性相关的细胞内铅受体和靶点蛋白质。铅刺激HK-2细胞出现凋亡、转分化时,是否有PbBPs参与其中?起了什么作用?在肾小管上皮细胞凋亡、转分化细胞模型上分离鉴定PbBPs,将可能弄清楚铅致细胞凋亡、转分化的真正原因所在,明了细胞内的分子靶点。并有可能找到新的高亲和性的PbBPs,为开发铅蛋白质络合剂药物提供证据。本课题以出现明显凋亡、转分化的HK-2细胞为研究对象,采用铅亲和柱一金属螯合亲和层析、一维SDS-PAGE电泳分离、ESI-MS/MS液-质联用串联质谱鉴定混合蛋白质样品,免疫印迹法验证,这种功能蛋白质组学方法筛选与HK-2细胞凋亡、转分化相关的铅结合蛋白,用生物信息学方法分析铅结合蛋白在铅致HK-2细胞毒效应中的可能作用。本研究首次在体外观察到醋酸铅具有刺激人肾小管上皮细胞转分化作用;发现6个与铅高亲和性结合蛋白质。蛋白质结构、功能分析显示6个铅结合蛋白与铅致HK-2细胞凋亡、转分化有关;建立了一条新的高通量分离鉴定铅结合蛋白的技术路线。本课题共分三章进行研究:第一章不同剂量醋酸铅致人肾小管上皮细胞凋亡及碘化钾的拮抗作用目的:研究醋酸铅对人近端肾小管上皮细胞系（HK-2）细胞毒性、细胞凋亡剂量-反应关系及碘化钾的拮抗作用;醋酸铅对NF-κB、bcl-2蛋白质表达的影响及与细胞凋亡的关系。方法:体外培养HK-2细胞分为不同剂量染铅组:分别加入浓度为0、2.5、5、10、20、40μmol/L的醋酸铅;碘化钾干预组:加入终浓度为40mg/L的KI、37℃孵育30分钟后加入上述不同剂量的醋酸铅。染毒72h后分别用噻唑蓝（MTT）法测定各组细胞的存活率;用相差显微镜观察细胞形态改变;Annexin-V/PI双染法检测细胞凋亡以及Hoechst33258染色法、荧光显微镜观察细胞凋亡形态;用间接免疫荧光-流式细胞术检测NF-KB、bcl-2蛋白表达改变。结果:（1）使HK-2细胞存活率明显下降的醋酸铅最低浓度是10μmol/L,醋酸铅浓度越高,细胞存活率越低,存在剂量-反应关系（r=-0.878,p＜0.01）。（2）醋酸铅作用72h,HK-2细胞LC₅₀为15.68μmol/L;（3）从5μmol/L铅剂量组开始细胞形态有明显改变,HK-2细胞由正常的立方形、多角形向长梭形细胞转变,高浓度铅组有大量细胞变小、变园,直至细胞脱壁漂浮;（4）2.5μmol/L醋酸铅即可引起HK-2细胞凋亡率明显增加（p＜0.05）,细胞凋亡率在10μmol/L铅浓度达到高峰值,铅浓度再增加,细胞凋亡率不再相应增加。（5）Hoechst33258染色,5μmol/L铅组细胞核染色加深呈高亮蓝色、模糊、核明显固缩。10μmol/L铅组大量细胞核浓染、固缩、核碎裂。（6）HK-2细胞NF-κB、bcl-2蛋白表达强度有随着染铅剂量增加而降低的趋势,NF-κB与Bcl-2降低显著相关（r=0.71,p＜0.01）。（7）细胞凋亡率与NF-κB、Bcl-2蛋白表达呈明显负相关（r=-0.625,p＜0.01与r=-0.709,p＜0.01）。（8）各剂量铅组加40mg/L的KI,可使细胞存活率增加,LC₅₀上升了3.33倍,细胞凋亡率下降,细胞形态改变明显改善。结论:（1）醋酸铅对HK-2细胞的毒性存在明显剂量反应关系,低剂量醋酸铅（2.5μmol/L）即可引起细胞凋亡明显增加,细胞形态改变,高浓度（10μmol/L）出现细胞大量坏死;醋酸铅对HK-2细胞的LC₅₀为15.68μmol/L。（2）醋酸铅能下调HK-2细胞NF-κB、Bcl-2基因的表达,这可能是其诱导HK-2细胞凋亡的途径之一。（3）40mg/L的KI可部分拮抗铅对人肾小管细胞HK-2的损伤作用。第二章醋酸铅与碘化钾对人肾小管上皮细胞表型转化以及致纤维化标志基因表达影响目的:探讨醋酸铅是否可以诱导人肾小管上皮细胞表型转化及其可能机制以及碘化钾的影响。方法:体外培养人近端肾小管上皮细胞系（HK-2）细胞分为不同剂量染铅组:分别加入浓度为2.5、5、10μmol/L的醋酸铅,以0.0μmol/L醋酸铅组作为对照;碘化钾干预组:加入终浓度为40mg/L的KI、37℃孵育30分钟后加入上述不同剂量的醋酸铅。染毒72h后应用相差显微镜、间接免疫荧光-流式细胞术、逆转录-聚合酶链反应（RT-PCR）观察HK-2细胞形态、a-SMA和FN蛋白表达的改变,以及对致纤维化因子TGF-β1、CTGF的影响。结果:（1）对照组正常人肾小管上皮细胞为近似方形或卵圆形,呈鹅卵石样排列,细胞间紧密衔接,融合成单层。5μmol/l铅组可见部分细胞形状变长、呈梭形,细胞间分离生长。10gmol/l铅组,除如上5μmol/l铅组改变以外,尚有部分细胞边界欠清晰、少数细胞变小、变园。（2）间充质细胞标记a-SMA阳性细胞百分率在5、10μmol/L铅组分别为9.32%、6.98%,与对照组比较明显增加,差别有统计学意义（p＜0.01）。（3）细胞外基质（ECM）标志蛋白FN随着醋酸铅剂量的增加而增加,在5、10μmol/L组较对照组升高8.3、8.4倍,差别有统计学意义（P＜0.01）。（4）与对照组比较,5、10μmol/L醋酸铅组TGF-β1mRNA以及蛋白表达均较对照组明显增加,差别有统计学意义（P＜0.01）。（5） 2.5、5、10μmol/L醋酸铅组CTGFmRNA及蛋白质表达均较对照组明显增加,差别有统计学意义（P＜0.05或P0.01）。（5）相同剂量醋酸铅组加40mg/L的KI孵育,能在一定程度上抑制上述改变。结论:（1）醋酸铅可诱导肾小管上皮细胞转分化,其作用机制可能与TGF-β1、CTGF表达调高有关。（2） 40mg/l碘化钾可以部分抑制铅诱导的肾小管上皮细胞表型转化以及TGF-β1、CTGF表达。第三章人肾小管上皮细胞HK-2铅结合蛋白分离鉴定目的:筛选染铅人肾小管上皮细胞铅结合蛋白;分析铅结合蛋白在铅诱导HK-2细胞凋亡、转分化过程中的可能作用;探索一条新的高通量分离鉴定铅结合蛋白的技术路线。方法:（1）人肾小管上皮细胞系（HK-2）细胞分为5μmol/L染铅组、对照组,醋酸铅作用72h收获细胞。（2）用非变性裂解液裂解细胞获得水溶性蛋白质样品,用3kDa超滤管脱盐和去除EDTA。（3）制备铅亲和柱（Pb-NTA）。（4）用铅亲和柱-金属螯合亲和层析分离与铅有高亲和性的蛋白质。（5） 1D-SDS-PAGE电泳分离蛋白质。（6）蛋白质胶内酶解。（7）混合蛋白质肽段经ESI-Q-TOF鉴定,输出pkl文件。（8）将仪器输出的pkl文件输入数据库（NCBInr）,使用Mascot软件中的串级质谱数据搜索功能进行搜索。（9）对获得鉴定的蛋白质进行生物信息学分析。（10）对质谱鉴定出来的其中2个蛋白质HSP90、TRAP-1进行Western-bloting分析验证。结果:（1） 5μmol/L染铅组亲和层析蛋白样品在SDS-PAGE胶上分离出5条清晰的蛋白质条带,分子量大致在15-100kDa之间;对照组亲和层析蛋白样品分离出1条较清晰的蛋白质条带,位置与染铅组带1一致。（2） 5μmol/L染铅组鉴定出6个铅亲和蛋白:①gi|306891 90kDaheat shock protein,HSP90;②gi|1082886 tumor necrosis factor type 1receptor associated protein,TRAP-1;③gi|13540513 oxysterol bindingprotein 2 isoform a,OSBP;④gi|3820484 ataxin-2-like protein,A2LP;⑤gi|119573321 G patch domain containing 4,isoform CRA b,GPATC4;⑥gi|60594178 Trex2 3’Exonuclease Structure,Trex2。对照组鉴定出2个与染铅组相同的铅亲和蛋白即:HSP90、TRAP-1。（3）蛋白质亚细胞定位、结构和功能分析:HSP90、TRAP-1、Trex2为金属结合蛋白,OSBP空间构象中具有负电荷聚集区,GPATC4、A2LP没有发现特定的金属结合位点;亚细胞定位:4个为细胞质蛋白质,其中一个线粒体蛋白。1个细胞核内蛋白质,1个核外在膜蛋白质HSP90、TRAP-1蛋白主要是细胞抗凋亡信号通路中的分子伴侣,协同其顾客蛋白起到抗凋亡作用。Trex2、A2LP、GPATC4涉及DNA损伤修复。OSBP与氧类固醇运输、解毒,与波形蛋白（vimentin）的聚集、解聚有关。HSP90可能是TGF-β/Smads信号通路的协同伴侣。（4）免疫印迹显示二组均有HSP90、TRAP-1表达,但染铅组表达量明显高于对照组。结论:（1）在凋亡、转分化HK-2细胞模型中筛选出6个铅结合蛋白;（2）铅诱导HK-2细胞凋亡、转分化可能与这些蛋白质与铅结合后,干扰这些蛋白质抗凋亡、DNA修复、调控转分化信号通路的功能有关。（3）铅亲和柱金属螯合亲和层析结合一维SDS-PAGE电泳、液-质联用串联质谱可以一次分离多个与铅有特异性、高亲和性结合的铅结合蛋白。更多还原

【Abstract】 Since the lead pollution of environment is common and the kindney is sensitive to lead,the disservice of nephrotoxicity of lead is universal both at home and abroad at present.The toxic effect of Lead on kidney is an insidious and progressive pathological process,and the early effct is subclinical and reversible,but it will turn out inreversible if devolops to a certain stage.Investigations show that the main target site in kidney of lead toxic effect is proximal tubules.The early pathological changes of lead nephrosis（LN） are mainly the formation of inclusion bodies in renal tubular epithelial cells and the damage of renal tubular epithelial cells, then renal interstitial fibrosis,renal tubule atrophy and loss of nephron gradually develop,finally lead to renal dysfunction.However,only a little has been known about the damage mechanism of renal tubular epithelial cells,which are the main target renal cells of lead,also there is a lack of systematic researchs on molecular toxicological mechanisms of LN.in tthis study,We observe apoptosis and epithelial-mesenchymal transdifferentiation（EMT） of human proximal tubular epithelial cell line（HK-2） by lead acetate inducing and the dose-response relationship of cytotoxic effects of lead acetate（Pb） to human proximal tubular epithelial cell.as well as the effect of lead acetate on the molecules that related to apoptosis and EMT signaling pathway,and the antagonistic effects of potassium iodide（KI）.And then,we employ the functional proteomics methods to isolate and identify the high affinity lead-binding proteins,try to explore the real mechanism of lead-inducing apoptosis and EMT of HK-2.Previous investigations had shown that the intracellular lead could interact with proteins of specific and high affinity and form the lead-binding proteins（PbBPs）.PbBPs are defined as the intracellular lead-receptors and target proteins with high affinity capacity of banding lead concerned with reactiveness and adaptability of cells.Are there PbBPs to being involved in the processes of HK-2 apoptosis and EMT by lead? What’s the role of PbBPs in these processes? For this reason,we had to isolate and identify PbBPs in the human renal tubular epithelial cell apoptosis and EMT model.In this way we could realize the real mechanism of lead-inducing apoptosis and phenotype transformation and identify the intracellular molecular targets,and might be able to identify a new high affinity PbBPs and provide evidence for exploration of protein chelating agent.lead affinity column-metal chelate affinity chromatography, one-dimensional SDS-PAGE electrophoresis and ESI-MS/MS liquid chromatography tandem mass spectrometry were used to isolate and identify In HK-2 showing obviously apoptosis and EMT,in this study.and immunoblotting was used to verify the PbBPs.Then bioinformatics was used to analyze the potential role of PbBPs in apoptosis and EMT of HK-2.The study is the first time observation EMT in HK-2 by lead acetate inducing in vitro;six high affinity lead-binding proteins involved in lead-inducing apoptosis and EMT in HK-2 were isolated and identified; A new high-throughout technical method to isolate and identify PbBPs had be set up.The study was divided into three parts:PartⅠThe apoptosis in HK-2 mediated by lead and the antagonistic effect of potassium iodideObjective:To investigate the dose-response relationship of cytotoxic effects of lead acetate（Pb） to human proximal tubular epithelial cell and the antagonistic effects of potassium iodide（KI）.And to explore the molecular mechanism involved in Pb inducing apoptosis. Methods:human proximal tubular epithelial cell line（HK-2） were divided in two groups.（1）treated with Pb（0,2.5,5,10,20,40μmol/L）.（2）pretreated with Pb（0,2.5,5,10,20,40μmol/L） puls KI of final concentration 40mg/L.The cells viability was measured with MTT assay and the cell morphology changes was observed with phase-contrast microscopy.The apoptosis was monitored by using flow cytometry and Hoechst 33258 staining with fluorescence microscopy.NF-κB,bcl-2 protein expression quantity was detected by flow cytometry.Results:（1） The minimum concentration of lead acetate which decreased The viability of HK-2 cells was 10μmol/L.The viability of HK-2 cells was negatively correlated with dosages of Pb（r= -0.878, P＜0.01） after exposure of Pb for 72h..（2） The concentrations of Pb that inhibited 50%(IC₅₀) of HK-2 cells at 72h was 15.68μmol/L.（3） HK-2 cells cultured in high dosages of Pb showed obvious morphologic changes,including elongated,contracted and rounded even agglomerated （4） apoptosis ratio was elevated significantly（p＜0.05）.（5） chromatin was condensed and cell nuclei exhibited the dense fluorescence in every dosages of Pb.（6） Pb also decreased NF-κB and Bcl-2 protein expression,Significant differences of NF-κB protein expression was found between 0μmol/L and 10μmoI/L,and of Bcl-2,were 0μmol/L and 5μmol/L and 10μmoI/L.（7） Correlation analysis showed that the rates of apoptosis were inversely correlated with the expression of NF-κB and Bcl-2(r= -0.625,p＜0.01;r=-0.709.（8） Compared with same dosages of Pb group,after pretreatment with KI,cell viability was increased,IC₅₀ being enhanced 3.33 times.Morphologic changes was significantly improved and apoptosis ratio was lowered.Conclusions:（1） There was a dramatically dose-response relationship between dosages of Pb and the toxic effects to HK-2 cells,. The low-dose lead acetate（2.5μmol/L） could cause significant apoptosis and cell morphology changes,while the high dose（10μmol/L） caused high degree of cells necrosis,and LC₅₀ of lead acetate in the HK-2 cells is 15.68μmol/L;（2） Pb induces apoptosis by inhibiting NF-κB and Bcl-2 expression in human proximal tubular epithelial cell line HK-2.（3） 40 mg./L KI can protect Pb-induced cytotoxicity in part.PartⅡChanges in expression of fibrotic markers and phenotype transformation in HK-2 exposed to lead acetate and potassium iodideObjective:To investigate whether lead acetate could induce human renal proximal tubule epithelial cells phenotype transformatation,and the possible mechanism,as well as the impacts of potassium iodide.Methods:human renal proximal tubule epithelial lines（HK-2） were cultured for 72 hours in different conditions as（1） treated with lead acetate（0,2.5,5,10μmol/L）.（2）treated with lead acetate（0,2.5,5,10μmol/L） puls KI（40mg/L）.The cell morphology changes was observed with phase-contrast microscopy,the expression of a-SMA,FN,TGF-β1and CTGF were assessed by immunofluorescence-flow cytometry.RT-PCR. was performed to test the expression of TGF-β1and CTGF mRNA.Results:（1） HK-2 cells in the control group were approximately of square or oval shape in a cobblestone-like arrangement way,and the cells connected to each other closely integrating into a monolayer.Some cells in the 5μmol/l lead group became longer and fusiform and grew separately.Besides the changes similar to 5μmol/l lead group,in 10μmol/L lead group,some cells’ borderlines were not clear,and a few cells shrunk and turned rounder.（2） Compared with the control group,the rate of the tubular epithelial cells expressing a-SMA significantly increased in 5、10μmol/L lead group（p＜0.01）.（3） The marked proteins FN in extracellular matrix（ECM） increased as lead acetate dose increased,and compared with the control group,the quantity of 5,10μmol/l lead groups increased to 8.3,8.4 times respectively（P＜0.01）.（4）Compared with the control group,the TGF-β1,CTGFmRNA and protein expression of HK-2 cells increased significantly（5、10μmol/L lead groups,P＜0.05）.（5） treating with lead acetate（0,2.5,5,10μmol/L） puls KI（40mg/L）,the changes described above can be inhibited to some extent. Conclusions:lead acetate can induce tubular EMT.TGF-β1/CTGF. Signaling pathway mediates the lead-induced EMT.Part Three:The isolation and identification of the lead-binding proteins in human renal tubular epithelial cells HK-2Objective:To screen the lead-binding protein in the lead-exposed human renal tubular epithelia cell HK-2,analyze the potential effects of lead-binding protein in the processes of lead-inducing cell apoptosis and EMT,and explore a new high-throughout technical method to isolation and identification of the lead-binding proteins.Method:（1） human renal proximal tubule epithelial lines（HK-2） were incubated with lead acetate for 72 h in twe conditions as treated with 0μmol/L and 5μmol/L lead acetate.（2） the cells were lysed with nondenaturing lysis buffer,then they were desalted and got rid of EDTA with 3 kDa ultrafiltration.（3） Prepared lead affinity column.（4） Isolated lead-binding proteins with lead affinity column metal chelate affinity chromatography.（5） Separated proteins with 1D-SDS-PAGE.（6） Enzymaticly hydrolyzed the proteins in gel.（7） identited the mixed protein peptides with ESI-Q-TOF,and the pkl document were outputted by the equipment.（8） searched protein database（NCBInr） with Mascot software.（9） proteins bioinformatics Analysis（10） confirmed proteins with Westein-bloting.Results:（1） There were 5 clear bands in treated with 5μmol/L lead acetate HK-2 protein sample on SDS-PAGE gel,while the control group protein sample,only one clear band.（2） 6 lead-binding proteins were identited from the HK-2 treated with 5μmol/L lead acetate.②gi|306891 90kDa heat shock protein, HSP90;②gi|1082886 tumor necrosis factor type 1 receptor associated protein,TRAP-1;③gi|13540513 oxysterol binding protein 2 isoform a, OSBP;④gi|3820484 ataxin-2-like protein,A2LP;⑤gi|119573321 G patch domain containing 4,isoform CRA_b,GPATC4;⑥gi|60594178 Trex2 3’ Exonuclease Structure,Trex2.And two lead-binding protein were identited from the control group,ie HSP90、TRAP-1.（3） structure and function of Proteins:HSP90,TRAP-1,Trex2 belong to metal-binding proteins.OSBP contains a negative-charge concentrating domain in space conformation,and no specific lead-binding sites were found in GPATC4,A2LP.Subcellular Localization:There were three cytoplasmic proteins,one mitochondrial protein,one nuclear protein, one external membrane protein.HSP90,TRAP-1 are the main molecular chaperones in the anti-apoptotic signaling pathway.and Trex2,A2LP, GPATC4 are involved in damaged-DNA repair.and OSBP is related to oxysteroid transport,detoxification,and the aggregation of the vimentin.then HSP90 might be a collaborative chaperone of the TGF-β/Smads signaling pathway.（4） westein-bloting showed that the protein expression of HSP90 and TRAP-1 in lead-treated group was higher compared with the control group.Conclusions:（1） Six high affinity lead-binding proteins were identited from HK-2 cell.（2） lead-inducing apoptosis and transdifferentiation in HK-2 may concerned with lead-bind proteins interfering the processes of anti-apoptosis,damaged-DNA repair and transdifferenation signal pathway.（3） The method of lead affinity column metal chelate affinity chromatography combining with 1D -SDS-PAGE and liquid chromatography-tandem mass spectrometry can isolate and identity multitude specific lead-binding proteins.更多还原