【作者】 覃保瑜；

【导师】 夏宁；

【作者基本信息】 广西医科大学 ， 代谢与内分泌， 2011， 博士

【摘要】 目的:1、运用“鸟枪法”(shotgun)蛋白质组策略分别对糖尿病大鼠在成模前、后的正常与肾病早期这两阶段的尿液作蛋白质组分析鉴定,并为探寻糖尿病肾病(DN)新的尿蛋白标志物及探讨DN发病机制提供实验基础。2、通过比较正常与早期DN大鼠肾皮质的氧化应激和线粒体功能相关指标、PGC-lαmRNA表达的变化等,探讨线粒体在DN发生和发展中的作用。3、运用双向荧光差异凝胶电泳(2D-DIGE)联合MALDI-TOF-MS质谱技术对正常与DN大鼠肾皮质的线粒体蛋白质作差异蛋白的分析鉴定,从亚细胞蛋白质水平探讨DN的发病机制。方法:以链脲佐菌素(STZ)诱发的Wistar大鼠糖尿病模型作为研究对象。1、分别收集大鼠(n=7)制模前后正常对照期和早期DN的尿液标本,标本经超滤管超滤浓缩、用层析柱去高丰度蛋白、去除杂质等处理后,蛋白样品经1-DE(SDS-PAGE)初步分离,随后胶内酶解蛋白、毛细管高效液相色谱分离、LTQ-Velos质谱设备作质谱分析(MS/MS)、蛋白质数据库搜索,鉴定蛋白质。对成功鉴定的蛋白质用生物信息学技术作细胞定位、功能组别、生物学过程及信号通路分析,对感兴趣的早期DN大鼠的特异表达蛋白质作更深入数据库检索及分析,寻找DN新的尿蛋白标志物及探讨DN发病机制。2、正常对照组(n=6)和早期DN组(n=7)大鼠处理后取肾皮质,用化学比色法检测氧化应激指标超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量以及线粒体功能指标琥珀酸脱氢酶(SDH)活性,实时荧光定量RT-PCR检测PGC-lαmRNA表达,比较2组间上述指标的变化。3、另外随机选取3只正常大鼠,运用密度梯度差速离心法对肾皮质的线粒体分别作粗提取和高纯质提取,同时提取肾皮质组织蛋白备作对照。电镜观察比较粗提取和高纯质提取线粒体的富集度、完整性,并作纯度初步鉴定;用Western blot方法比较肾皮质粗提线粒体、高纯质线粒体和组织蛋白中胞浆成分标志蛋白GAPDH的表达,鉴定提取线粒体蛋白的纯度。提取正常对照组和早期DN组大鼠肾皮质的高纯质线粒体,裂解线粒体提取蛋白、去除杂质,随后以蛋白样品作双向荧光差异凝胶电泳(2DE-DIGE),软件分析两组间差异蛋白点后,作制备胶,切胶取差异显著的蛋白点,胶内酶解,MALDI-TOF-MS质谱分析,蛋白质数据库检索鉴定差异蛋白质。结果:1、糖尿病前、后两阶段的尿蛋白经shotgun分析和数据库检索,共成功鉴定了218个蛋白质,从中结果筛选出11个DN早期的候选诊断标志蛋白,利用生物信息学方法分析蛋白质的细胞定位、功能组别、生物学过程等。进一步对这11个候选标志蛋白生物学通路(KEGG-PATHWAY)分析结果发现,Alpha-actinin-4参与粘着斑通路、紧密连接通路、粘附连接通路、白细胞经上皮移行通路等多个信号通路。其中粘着斑通路涉及细胞外基质(ECM)和生长因子(GF)诱导的粘附斑激酶(FAK)信号转导通路、磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号转导通路、丝裂原激活的蛋白激酶(MAPK)信号传导通路、RhoA-ROCK信号转导通路等。结合生物信息学分析和文献检索发现:Protein S100-A8和C-reactive protein也与DN的发生发展有关,这2个蛋白和Alpha-actinin-4可能成为研究DN发病机制标志蛋白。2、与正常对照组比较,早期DN组大鼠肾皮质的丙二醛(MDA)量增高,超氧化物歧化酶(SOD)和琥珀酸脱氢酶(SDH)活性降低;实时荧光定量RT-PCR检测过氧化物酶体增殖物激活受体γ辅激活因子lα(PGC-lα)mRNA表达结果显示,与正常对照组比较,早期DN组大鼠肾皮质的PGC-lαmRNA表达下降。3、电镜结果显示,与粗提取线粒体比较,高纯质提取线粒体富集度和纯度更高;两种方法提取的线粒体均包膜完整。Western blot结果显示,与组织提取蛋白比较,粗提取线粒体蛋白中有极少量的胞浆成分标志蛋白GAPDH的表达,而高纯质线粒体蛋白中无GAPDH的表达。4、2DE-DIGE结果显示,分别成功分离大鼠肾皮质的线粒体蛋白(357±32)个蛋白点,两组间差异>3.0倍的显著差异蛋白点13个,经MALDI-TOF-MS质谱分析和蛋白质数据库检索,成功鉴定8个蛋白质,这些蛋白质在早期DN组表达均显著降低,生物信息学分析发现这些蛋白质与氧化磷酸化及线粒体内的氨基酸和脂肪酸代谢有关,提示早期DN均可能存在三大营养物质的代谢障碍。结论:1、shotgun蛋白质组技术具有高效、快速及较高通量的特点,适合用于如尿液这种含较多盐分和杂质的比较复杂的标本蛋白质研究。蛋白质组技术能全景式的分析尿液蛋白质,有助于研究者较快的从尿液中发现DN相关的标志蛋白。2、用shotgun技术跟踪分析糖尿病大鼠患病前后不同阶段的尿液蛋白质组变化,此法目前在国内外均鲜有报道。本研究筛选出11个DN早期候选诊断标志蛋白,结合生物信息学分析方法和文献检索发现:Protein S100-A8、C-reactive protein、Alpha-actinin-4可能成为研究DN发病机制的标志蛋白。3、本研究中,DN早期大鼠肾皮质的SOD活性降低, MDA含量升高,说明DN与氧化应激密切相关。同时检测肾皮质SDH活性以及PGC-lαmRNA表达下降,提示DN的发病与线粒体的功能紊乱及生物合成障碍有关。有关DN与PGC-lα关系的研究目前国内也外均鲜有报道,值得深入探讨。4、本研究对早期DN和正常大鼠肾皮质线粒体作2DE-DIGE差异蛋白分析后,成功鉴定8个在早期DN组表达差异显著降低的蛋白,这些蛋白质与线粒体氧化磷酸化、氨基酸和脂肪酸代谢有关,提示了DN发生发展与线粒体有关,具体机制值得进一步研究。更多还原

【Abstract】 Objective:1.To identify and analyse urine proteins between normal stage when the models have been made or not and the early stage of kidney disease when the models have been made in diabetic rats separately with“shotgun”which is a study strategy of proteomics. By doing this we can provide fundamental experimental basis to search for new urinary protein markers and investigate the pathogenesis of diabetic nephropathy.2.To investigate the role of mitochondria in the occurrence and development of diabetic nephropathy through comparing the related indicators of renal cortical oxidative stress and mitochondrial dysfunction and the change in PGC-lαmRNA expression between normal and the early stages of diabetic nephropathy rats.3.To investigate the pathogenesis of diabetic nephropathy at the sub-cellular proteins level by analyzing and identifying differential proteins of renal cortical mitochondria in normal rats and diabetic nephropathy rats through two-dimensional fluorescence difference gel electrophoresi(s2D-DIGE)as well as MALDI-TOF-MS.Methods:The research objects are diabetic Wistar rat models which are induced by streptozotocin (STZ).1.Collect urine specimen of normal control period when the models have been made or not and the early stage of kidney disease when the models have been made separately from 7 rats. After ultrafilter concentration, depletion of high abundant proteins by chromatographic column and removing impurity we preliminarily separated the protein samples by 1-DE(SDS-PAGE). Then enzymatic hydrolysis of proteins in the glue, capillary high performance liquid chromatographic separation, mass spectrum analysis by LTQ-Velos and searching protein databases have been done to identify proteins. Use bioinformatics technology to study the cellular localization, functional groups, biological process and signaling pathways of proteins which have been identified. In order to search for new urinary protein markers and investigate the pathogenesis of diabetic nephropathy we search the data base further to analyze specific proteins which we are interested in among rats of the early stage of kidney disease.2.Take renal cortical separately from normal controls(n=6) and the early diabetic nephropathy group(n=7) of rats to compare indexes such as superoxide dismutase (SOD) activity which is an oxidative stress index and can be detected by chemical colorimetric method, the content of malondialdehyde (MDA) ,the activity of amber acid dehydrogenase (SDH) which is a mitochondrial function index and the expression of PGC-lαmRNA which can be tested by Real-time fluorescence quantitative RT - PCR detection.3.Extract high purity and crude extract of renal cortical mitochondria and renal cortical tissue proteins by velocity centrifugation method from three normal rats which are chosen by random. Compare the concentration and integrity between high purity and crude extract of mitochondria and identify purity of mitochondria preliminarily by electron microscope. Compare GAPDH which is a biomarker protein of cytoplasm among high purity and crude extract of mitochondria and histones. Identify the purity of the mitochondrial protein. Extract high purity of renal cortical mitochondria from normal controls and the early diabetic nephropathy group of rats. Lyse mitochondria to extract proteins and meanwhile remove impurities. Then identify different proteins by two-dimensional fluorescence difference gel electrophoresis(2D-DIGE), software analysis, glue preparation, cutting glue, enzymolysis, MALDI-TOF-MS technique and searching in proteins database.Results:1. There are 274 proteins which are identified by shotgun analysis and database search among urine proteins during the two stages, 121 of them are proteins during the normal stage and 153 of them are proteins during the early stage of diabetic nephropathy. There are 56 urine proteins which are the same during the two stages. There are 65 urine proteins uniquely during the normal stage and 97 urine proteins uniquely during the early stage of diabetic nephropathy. Analyze cellular localization, functional groups, biological process, signaling pathways and so on by bioinformatics approach. Analyze biological pathway of 97 proteins during the early stage of diabetic nephropathy and then select one proteins which called Alpha-actinin-4.This proteins have been proved to be related to focal adhesion kinase(FAK ) signal transduction pathway which is induced by extra cellular matrix(ECM)and growth factor(GF), phosphatidyl-inositide 3 kinase/AKT (PI3K/AKT) signal transduction pathway , protein kinase signal transduction pathway which is activated by mitogen and RhoA-ROCK signal transduction pathway.2.Compared to normal stage, the levels of malondialdehyde (MDA) is higher while activity of superoxide dismutase (SOD) and succinate dehydrogenase (SDH)is lower in the renal cortex of rats during the early stage of diabetic nephropathy. The real-time fluorescence quantitative RT - PCR detection has proved the expression of PGC-lαmRNA in the renal cortex of rats during the early stage of diabetic nephropathy is lower than normal.3.Being observed under electron microscope, both high purity and crude extract of mitochondria have intact membranes and the concentration and purity is higher in high purity of mitochondria. Compared with organization extraction protein, crude extract of mitochondria shows extremely few GAPDH biomarker proteins of intracellular compositions while high purity of mitochondria shows none of it by Western blot.4. 357±32 spots have been isolated successfully from the high purity of renal cortical mitochondria samples by two-dimensional fluorescence difference gel electrophoresis(2D-DIGE).There are 13 spots where differences between the two groups are greater than three times. We have successfully identified 8 proteins by MALDI-TOF-MS.,which are lower during the early stage of diabetic nephropathy ,The proteins link to oxidative phosphorylation and amino acid and fatty acid metabolism in the mitochondria suggest there may be metabolic disorders of three macronutrients during the early stage of diabetic nephropathy by bioinformatics methods.Conclusion:1.Shotgun is an efficient, rapid, good detection limit and high flux proteomic technique, which is suitable for protein analysis of complex specimens which are rich for salinity and impurity such as urine. Proteomic technique may help researchers to analyze urine proteins panoramically, which is helpful for find out diabetic nephropathy relevant biomarker proteins more quickly from urine.2.Following and analyzing the proteome changes of urine by shotgun technology during the different stages of disease in diabetic rats has been seldom reported both at home and abroad. In this study, we found that Alpha-actinin-4 appear on the early stage of diabetic nephropathy and do with several important signal transduction pathways link to diabetic nephropathy. These findings suggest that this proteins can not only be candidate markers of early diagnosis but also be biomarker proteins for the study on the mechanisms of diabetic nephropathy.3. In this study, SOD is less active and MDA levels rise, which confirm the perspective that diabetic nephropathy is closely connected to oxidative stress again. At the same time, SDH is less active and PGC-lαmRNA levels drop, which suggest that diabetic nephropathy is related to the dysfunction and synthesis obstacles of mitochondria. Relations between diabetic nephropathy and PGC-lαhas been seldom reported both at home and abroad, which need to do more research.4.Two-dimensional fluorescence difference gel electrophoresis(2D-DIGE)has been used to analyse different proteins among the early stage of diabetic nephropathy and normal stage of rats in the study, which identify 8 different proteins that substantially decrease. These proteins are related to mitochondrial oxidative phosphorylation, acid metabolism and fatty acid metabolism, which suggest that diabetic nephropathy is connected to mitochondria and the mechanism requires further researching.更多还原