【作者】 陈书尚；

【导师】 孙颖浩； 王林辉； 许传亮； 侯建国；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 一、研究目的1、通过观察不同结石成分肾结石患者肾乳头的组织病理学特点和钙盐沉积特点,分析局部病理改变与钙盐沉着在肾结石形成中的作用;通过检测成骨性蛋白骨桥蛋白、骨形成蛋白-2(BMP-2)和Ⅱ型胶原在肾结石患者肾组织的表达,以验证我们关于。肾结石患者肾组织钙盐沉积的形成可能也是一种成骨性反应的猜测,探讨肾钙盐沉积的机制;2、通过观察不同浓度草酸、COM结晶对体外培养的人近端肾小管上皮细胞(HK-2)的毒性损伤和蛋白表达的影响,探讨上皮细胞受损在肾结石形成过程中的可能作用;3、通过双向凝胶电泳结合质谱分析,鉴定HK-2细胞在受到草酸、COM结晶损伤后表达的差异蛋白质,并推测和讨论这些蛋白质在HK-2细胞受损和肾结石形成中的可能作用,为进一步深入研究肾结石的形成机制提供新的研究思路和方向。二、研究内容1、采用傅立叶转换红外光谱测定结石成分;对接受经皮肾镜取石碎石术(PCNL术)的肾结石患者,于术中直视下获取肾乳头活检标本,行HE染色和茜素红染色,光镜和电镜下观察其组织病理学特点;采用免疫组化检测骨桥蛋白、BMP-2和Ⅱ型胶原在肾结石患者肾组织的表达情况,并与正常肾组织比较。2、体外培养HK-2细胞,待细胞长满后,换成无血清含钙DMEM培养液,使细胞处于静止期;分别在培养基中加入不同终浓度(1 mM、2 mM、3 mM、5 mM和10mM)草酸、(200mg/L、500mg/L和1000mg/L)COM结晶或(1 mM、2 mM、3 mM、5 mM和10mM)草酸+200mg/L COM结晶,作用4h、12h及24h后,以CCK-8试剂盒检测上述结石成分对HK-2细胞的毒性作用;通过Bradford蛋白浓度测定试剂盒检测HK-2细胞受不同浓度(1 mM、2 mM、5 mM和10mM)草酸或(1mM、2mM、5mM和10mM)草酸+200mg/L COM结晶作用后,细胞表达的总蛋白量的变化。3、筛选受草酸和COM结晶损伤后HK-2细胞的差异蛋白表达谱:利用双向凝胶电泳技术对正常HK-2细胞和与2mM草酸+200mg/L COM结晶共孵育12小时后的HK-2细胞总蛋白进行电泳分离,获得相应蛋白质谱。Image Master Platinum5.0图形分析软件对凝胶图谱进行分析,筛选出正常HK-2细胞和与草酸+COM结晶孵育后细胞间的差异蛋白质点。4、使用Finnigan线性离子阱串联质谱仪(LTQ)对部分差异表达蛋白点进行液相色谱—串联质谱分析(LC-ESI-MS/MS);从所鉴定出来的差异蛋白点选择2种蛋白ENO1和Cofilin-1,用Western blotting验证其在正常HK-2细胞和受损HK-2细胞中的表达情况;通过检索数据库,对所鉴定出来的蛋白质进行亚细胞定位和功能分析,并对这些蛋白在肾小管上皮细胞受损和肾结石形成中的可能作用进行推测和讨论。三、结果1、部分肾结石患者的肾脏可见肾乳头钙斑;活检标本的组织病理学检查发现局部有钙盐沉积。钙盐沉积的镜下特点:在钙盐聚集较多的组织切片,钙盐镜下表现为结节状或片状,可见于肾小管基底膜和间质组织;而在钙盐较少的组织标本,钙盐仅见于肾小管基底膜,或呈颗粒状由肾小管基底膜向间质蔓延;当钙盐突破尿路上皮进入集合系统后,其上可见小结石生长。免疫组织化学检查表明:肾结石患者肾组织中的肾小管上皮细胞可见骨桥蛋白呈阳性表达,而肾结石患者肾组织和正常肾组织均未见BMP-2和Ⅱ型胶原明显表达。2、细胞毒性检测表明,草酸和/或COM结晶对HK-2的毒性作用呈明显浓度依赖性,但在不同的浓度与作用时间对HK-2作用不同。蛋白含量检测表明:1mM、2mM和5 mM草酸作用12h后,HK-2表达的蛋白量增加,但无统计学意义(P均＞0.05);而受10mM草酸或10mM草酸+200mg/L COM结晶作用12h后,HK-2表达的蛋白量则显著减少(分别为263.75±75.77ug/ml vs 328.75±60.34ug/ml,227.5±54.97ug/ml vs328.75±60.34ug/ml,P＜0.05);2mM草酸+200mg/L COM结晶作用12h后,HK-2表达的蛋白量稍高于正常HK-2细胞,但无统计学意义(332.5±51.48ug/ml vs328.75±60.34ug/ml,P＞0.05)。3、成功建立正常HK-2细胞和受2mM草酸+200mg/L COM结晶作用12小时后的HK-2细胞总蛋白的双向凝胶电泳图谱,实验组凝胶最后共得到2204±84个蛋白点,正常细胞组即对照组最后共得到2347±57个蛋白点。经软件分析,实验组凝胶与对照组凝胶比较共找到可信差异表达蛋白点16个。其中实验组表达上调的有9个,表达下调的有7个。4、差异蛋白点利用LC-ESI-MS/MS技术进行了质谱鉴定,最后共有12个蛋白质点得到了鉴定。有许多研究表明这些蛋白参与了细胞的能量代谢、细胞增殖、凋亡、钙离子通道活性调控、细胞运动及信号转导等多种生理功能。Western blotting检测证实了受损HK-2细胞中ENO1蛋白表达升高,而Cofilin-1表达降低。四、结论1、肾结石患者钙盐沉积最初可能形成于肾小管基底膜,并向间质扩展。钙盐在肾结石形成中至少起到了以下作用:①钙盐突破上皮进入集合系统,可作为结晶粘附、聚集和生长的平台;②脱落到集合系统的钙盐可作为结石形成的核心;③钙盐与局部细胞相互作用,影响肾结石的形成。2、肾结石患者肾组织表达骨桥蛋白,而不表达BMP-2和Ⅱ型胶原,因此钙盐的形成可能并不是一种类似于动脉钙化的成骨性反应。3、草酸、COM结晶在不同的浓度与作用时间对HK-2细胞产生不同程度的毒性作用。4、高浓度草酸和COM结晶可使正常人HK-2细胞的蛋白表达发生改变,这些蛋白的功能涉及能量代谢、细胞增殖和凋亡、细胞运动、钙离子通道活性调节、信号转导、蛋白合成调节和细胞应激反应等多方面,既可起到细胞的自我保护作用,又可能通过相应途径在肾结石的形成过程中起重要作用,对这些蛋白与肾结石关系的进一步探讨,有望发现影响肾结石形成的新的作用途径和机制5、本研究将双向凝胶电泳结合蛋白质谱分析应用于尿路结石的基础研究,为了解尿石结晶与上皮细胞的相互作用、研究肾结石形成机制和寻找预防肾结石发生和复发的有效措施提供了新的方法和研究方向。更多还原

【Abstract】 Objective1.To observe the histopathological characters of renal papilla and renal calcareous deposits in patients with various renal calculi,so as to explore the possible role of local histopathological changes and calcareous deposits in the formation of renal calculi.To explore the formation mechanism of renal calcareous deposits by determining the expression of osteopontin,bone morphogenetic protein-2（BMP-2） and typeⅡcollagen in the kidneys of patients with renal calculi.2.To evaluate the toxic effects of oxalic acid and/or calcium oxalate monohydrate （COM） crystals at different levels on human renal tubular epithelial cells（HK-2） as well as the influence on cell protein expression;to explore the potential role of epithelial cell injury in the formation of renal calculi.3.To identify the differently expressed proteins in HK-2 cells injured by oxalic acid and COM crystals,and to postulate and discuss the potential roles of these proteins in renal tubular epithelial cell injury as well as kidney stone formation,so as to provide new ways for further kidney stone formation.Methods1.Patients with renal calculi undergoing percutaneous nephrolithotomy（PCNL） were included,whose stones were analysed with a transform-infrared（FT-IR） spectrometer. Renal papilla biopsy specimens were obtained under a nephroscope during the operation, followed by staining with hematoxylin-eosin or alizarin bordeaux for light microscopy and electron microscopy to observe the histopathological characters.The expression of osteopontin,bone morphogenetic protein-2（BMP-2） and typeⅡcollagen in the kidneys of patients with renal calculi or without urolithiasis were determined by immunohistochemistry.2.Normal HK-2 cells were cultured in vitro and the culture medium was changed with serum-free medium after cell growth to confluence.Oxalic acid and/or COM crystals with different concentrations were then added and the cells were incubated for 4h,12h or 24h,respectively.Crystal adherence to cells was observed microscopically.The toxic effects of oxalic acid and/or COM crystals with different concentrations on HK-2 cells after incubation for 4h,12h or 24h were detected with a CCK-8 kit.Changes of protein expression in HK-2 cells were determined using the Bradford method.3.The proteins of normal HK-2 cells and the HK-2 cells incubated with 2mM oxalic acid plus 200mg/L COM crystals for 12h were respectively separated using two-dimensional electrophoresis technology and visualized by silver staining.The digitized images were then analyzed with an Image Master software to obtain the differential protein profiling between normal HK-2 cells and the HK-2 cells incubated with oxalic acid plus COM crystals.4.The differential protein spots were identified using liquid chromatography-electrospray ionization-mass spectrometry/mass spectrometry（LC-ESI-MS/MS） conducted by a Finnigan LTQ mass spectrometer.Two identified proteins,ENO1 and Cofilin-1,were selected and their expression in normal HK-2 cells or injured HK-2 cells was determined by Western blotting.All identified proteins were classified by their subcellular location and biological function.The potential roles of these proteins in renal tubular epithelial cell injury as well as kidney stone formation were postulated and discussed.Results1.Renal papilla calcific plaques were found in a part of the patients with renal calculi during PCNL.Local calcareous deposits could be found in the biopsy specimens. Microscopically,in tissue sections with abundant calcareous deposits,calcareous deposits appear as tuberose or flakiness,localized in the tubular basement membranes and interstitial tissues;while in other sections,calcareous deposits could only be observed in the tubular basement membranes or spreading from the tubular basement membranes to the interstitium.Once the calcareous deposits pierced into the collection system,tiny stones growing on them could be observed.Immunohistochemistristry examination showed that osteopontin was positively expressed in the renal tubular epithelial cells of the patients with kidney stones and the normal controls,but BMP-2 and typeⅡcollagen were negatively expressed in the kidneys of both groups.2.Oxalic acid and/or calcium oxalate monohydrate presented a concentration-dependent toxic effect on HK-2 cells which was,however,not merely increased with time lasting.The quantity of protein expressed by HK-2 cells incubated with 10mM oxalic acid or 10Mm plus 200mg/L crystals was significantly smaller than that of control（263.75±75.77ug/L vs 328.75±60.34ug/L and 227.5±54.97ug/ml vs 328.75±60.34ug/ml,respectively,P＜0.05）.After incubated with 2mM oxalic acid plus 200mg/L COM crystals,HK-2 cells expressed more proteins than normal,but the different was not significant（332.5±51.48ug/ml vs 328.75±60.34ug/ml,P＞0.05）. 3.The two-dimensional gel electrophoresis were successfully performed for isolating the proteins of normal HK-2 cells（Control Group） or HK-2 cells incubated with 2mM oxalic acid plus 200mg/L COM crystals（Experiment Group）.Visualized spots were 2204±84 in the experiment group and 2347±57 in the control group.Software analysis revealed 16 protein spots showing differential expression between the two groups.In the experiment group,among these 16 protein spots,the up-regulated proteins were 9,while the down-regulated proteins were 7.4.Differential protein spots were identified using LC-ESI-MS/MS and analyzed by database searching.A total of 12 proteins were eventually identified,the function of which includes cellular energy metabolism,proliferation,apoptosis,Ca²⁺ channel activity regulation,cell movement and signal transduction.Western blotting confirmed that the injured HK-2 cells had higher expression of ENO1 but lower expression of Cofilin-1.Conclusions1.Renal papillary calcareous deposits in a part of patients with renal calculi may be initiated in the renal tubular basement membranes and spread into the interstitium.The role of renal calcareous deposits in kidney stone formation may include the following aspects:1) Calcareous deposits act as platforms for crystal adherence,aggregation and growth when piercing into the collection system;2) Detachment of calcareous deposits in the collection system may become the nucleus of stones;3) The interaction between calcareous deposits and local cells may be related to the formation of renal calculi.2.BMP-2 and typeⅡcollagen are negatively expressed in the kidney of patients with renal calculi,which suggests that the formation of renal calcareous deposits may not be an osteoblastic reaction resembling arteriosteogenesis.3.The toxic effect of oxalic acid and/or COM crystals on HK-2 cells was concentration dependent but is not merely increased with time lasting.4.High levels of oxalic acid and COM crystals can cause protein expression profile changes in normal human HK-2 cells.The function of concerned proteins includes energy metabolism,cell proliferation and apoptosis,cell movement,calcium channel activity regulation,signal transduction,protein synthesis and cellular stress reaction.The changes of protein expression may not only self-protect HK-2 cells from being injured,but also be related to kidney stone formation.Further studies on these proteins are required so as to find out new mechanism and pathways leading to stone formation.5.This study provides an important knowledge of oxalic acid and COM crystal-treated HK-2 cells’ protein expression in vitro and should be helpful for the further elucidatation of the molecular mechanisms involved in the interaction between urinary stone ingredient crystals and renal epithelial cells and kidney stone formation,and provide new methods and for seeking effective precautionary measures for the recurrence of renal calculi.更多还原