【作者】 颜玉蓉；

【导师】 邱宗荫；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2008， 博士

【摘要】 目的建立羟基喜树碱(Hydroxycamptothecin, HCPT)诱导的肝癌SMMC-7721细胞线粒体凋亡模型,应用蛋白质组学分析的电泳法策略和鸟枪法策略鉴定细胞凋亡前后的线粒体差异表达蛋白质和细胞核差异表达蛋白质,为从亚细胞器蛋白质组水平进一步阐明HCPT诱导肝癌细胞发生凋亡的机理奠定基础,同时建立并完善基于稳定同位素标记的亲和标签标记法(cICAT)的亚细胞器定量蛋白质组学技术平台。策略与方法1.确定肝癌细胞线粒体凋亡时相:用HCPT处理肝癌SMMC-7721细胞,通过光镜、MTT实验、电镜、流式细胞仪和AO/EB双染等方法分别在定性、定量、早期与晚期等不同层面上检测HPCT的致肿瘤细胞凋亡活性;通过线粒体跨膜电位、激光共聚焦与western blot法等检测线粒体凋亡的时相变化,从而确定线粒体凋亡时相。2.线粒体分离与纯度鉴定:用线粒体组分分离试剂盒分离肝癌SMMC-7721细胞线粒体,用詹纳斯绿B染料对分离的线粒体进行染色鉴定;分别用核组蛋白Histone、细胞骨架蛋白β-actin、组织蛋白酶Cathepsin-D和热休克蛋白HSP60作为细胞核、细胞浆、溶酶体和线粒体的特异标志蛋白,用western blot对分离的线粒体进行纯度鉴定;分别用琥珀酸脱氢酶作为线粒体纯度的指标、NADPH-细胞色素C还原酶作为内质网含量的指标、5-?核苷酸酶作为细胞膜含量的指标,通过测定酶比活力的方法对分离的线粒体进行纯度鉴定。3.应用双向电泳/基质辅助激光解吸离子化飞行时间质谱( two-dimensional electrophoresis/matrix assisted laser desorption/isonization time of flying mass spectrometry, 2DE-MALDI-TOF/MS)技术路线分析细胞凋亡前后的线粒体差异表达蛋白谱:通过优化各种条件参数来确定合适的样品上样量、染色方法与胶条种类;根据蛋白质溶解度不同将线粒体蛋白分成三个组分,分别对三个组份进行双向电泳分离,用PDquest(7.0)软件分析扫描的二维凝胶电泳图像,筛选出细胞凋亡前后的差异表达蛋白斑点;候选差异表达蛋白斑点经胶内胰酶酶解,MALDI-TOF/MS分析,得到肽指纹图谱(peptide mass fingerprinting, PMF),使用Mascot和PeptIdent软件分别在MSDB和SWISS-PROT数据库中检索鉴定差异表达蛋白质,并对差异蛋白质进行生物信息学分析。4.应用基于二维液相色谱/串联质谱(two-dimensional liquid chromatography/tandem mass spectrometry,2D-LC/MS/MS)的可裂解的稳定同位素标记的亲和标签(cleavable isotope-coded affinity tag,c-ICAT)方法定量分析细胞凋亡前后线粒体蛋白质组的表达差异:分别对线粒体中等溶解度蛋白组份和线粒体疏水蛋白组份进行c-ICAT定量蛋白质组分析。蛋白样品经过c-ICAT试剂标记、酶解、强阳离子交换色谱/亲和色谱纯化标记肽、裂解去除生物素亲和标签、μRP-HPLC MS/MS分析,根据肽的洗脱峰面积得到同一种蛋白质在HCPT促肝癌细胞凋亡前后的线粒体中表达量变化的准确信息、根据MS/MS分析结果进行测序获得到蛋白质的鉴定信息。并对表达量有显著差异的蛋白质进行生物信息学分析。5.细胞核纯度鉴定及细胞凋亡前后细胞核蛋白质组表达差异的定量分析:用细胞组分分离试剂盒分离获取肝癌SMMC-7721细胞核组份;分别用热休克蛋白HSP60、细胞骨架蛋白β-actin、核组蛋白Histone和组织蛋白酶Cathepsin-D作为线粒体、细胞浆、细胞核和溶酶体的特异标志蛋白,用western blot对分离的细胞核进行纯度鉴定。用c-ICAT试剂标记提取的核蛋白质,经过标记、酶解、色谱纯化、裂解等过程,采用μRP-HPLC MS/MS在线分析,根据定量积分肽的洗脱峰面积得到同一种蛋白质在HCPT促肝癌细胞凋亡前后的细胞核中表达量变化的准确信息、根据MS/MS分析结果测序获得到蛋白质的鉴定信息。并对表达量有显著差异的蛋白质进行生物信息学分析。结果1. HCPT对肝癌SMMC-7721细胞的增殖有明显抑制作用,半数抑制浓度(50% inhibitory concentration, IC50)约为80μg/ml。当用80μg/ml HCPT对细胞处理不同时间后,细胞可发生一系列形态学和生化性质的变化:细胞膜的磷脂酰丝氨酸外翻,细胞核染色质固缩、呈致密浓染的凋亡状态,超微结构观察发现线粒体出现了肿胀性变化,线粒体跨膜电位下降,并且Cytc从线粒体释放至细胞浆。2.电镜观察染色结果:分离的线粒体经詹纳斯绿B染色后,呈蓝绿色的颗粒状或棒状结构; western blot结果表明:提取的线粒体组分检测到了线粒体标志蛋白HSP60,没有检测到细胞核标志蛋白Histone、细胞浆标志蛋白β-actin与溶酶体标志蛋白Cathepsin-D;酶比活力测定结果显示:提取的线粒体组分与细胞匀浆相比较,琥珀酸脱氢酶比活力提高13.8倍,NADPH-细胞色素C还原酶和5-?核苷酸酶在线粒体组分中的比活力很低。3.优化条件试验确定:最合适的样品上样量为200μg、最佳染色方法为银染、最佳胶条为非线性pH3-l0胶条。通过对细胞线粒体凋亡前后的二维凝胶电泳图像进行对比分析,发现约有39个蛋白斑点表达有差异,对其中25个差异蛋白斑点进行MALDI-TOF/MS分析后,鉴定出了20种可能的差异表达蛋白。4.对细胞凋亡前后线粒体中等溶解度蛋白组份的c-ICAT分析结果显示:分析鉴定了91种蛋白质,获得了在细胞凋亡前后其相对表达量差异有显著统计学意义的蛋白质74种,其中,42种蛋白质在细胞凋亡后的表达量下调,32种蛋白质的表达量在凋亡细胞中上调。5.对细胞凋亡前后线粒体疏水蛋白组份的c-ICAT分析结果显示:分析鉴定了244种蛋白质,获得了154种蛋白质在细胞凋亡前后的相对表达量差异有统计学意义,其中,12种蛋白质在凋亡细胞中的表达量下调,137种蛋白质的表达量在凋亡细胞中上调,5种蛋白质的表达量没有变化。鉴定的蛋白质中有13种强碱性蛋白质,分子量大于200KDa的11种蛋白质和小于10KDa的6种蛋白质,50种膜蛋白质,表达量改变10倍以上的蛋白质有45种。6.提取的细胞核组分检测到了细胞核标志蛋白Histone,没有检测到线粒体标志蛋白HSP60、细胞浆标志蛋白β-actin与溶酶体标志蛋白Cathepsin-D。对细胞凋亡前后细胞核蛋白组份的c-ICAT分析结果显示:分析鉴定了94种蛋白质,获得了相对表达量差异有统计学意义的蛋白质43种,其中,12种蛋白质在细胞凋亡后的表达量下调,30种蛋白质的表达量在凋亡细胞中上调,1种蛋白质的表达量没有变化。结论1. HCPT可通过Cytc依赖的线粒体途径诱导癌细胞发生凋亡。2.三种线粒体纯度鉴定试验均说明:提取的细胞器组份是线粒体并且线粒体纯度较高,可用于后续的蛋白质组分析。3.采用2DE-MALDI-TOF/MS技术路线鉴定出的线粒体中20种差异表达蛋白可能在HCPT诱导的线粒体凋亡途径中起重要作用。4.细胞凋亡前后线粒体中等溶解度蛋白质组中表达量差异蛋白的分子功能主要与能量代谢、核酸的翻译、转录、复制以及细胞骨架有关。5.细胞凋亡前后线粒体疏水蛋白质组中表达量差异蛋白的分子功能主要与能量代谢,细胞结构,核酸代谢,核糖体,细胞分裂增殖、分化凋亡,以及信号转导有关。6.细胞凋亡前后细胞核蛋白质组中表达量差异蛋白的分子功能主要与细胞增殖、凋亡和分化,核酸代谢,细胞骨架,以及能量代谢有关。几种在凋亡细胞的线粒体和核中的表达量都上调的蛋白质,对HCPT的抗癌机理研究和细胞凋亡的分子机制研究有重要意义。这些发现不仅为从蛋白质水平进一步阐明HCPT诱导癌细胞发生凋亡的机理奠定了基础,也说明了本实验所建立的研究策略有助于亚细胞定量蛋白质组学研究和药物蛋白质组学研究。更多还原

【Abstract】 ObjectiveTo quantify and qualify the mitochondrial proteome and nuclear proteome in hydroxycamptothecin(HCPT)-treated hepatoma cells with gel-based proteomics strategy and shotgun proteomics strategy, for further elucidating the mechanism of HCPT-mediated cell apoptosis at subcellular proteomics level. Moreover, to set up a technical platform based on cleavable isotope-coded affinity tag(c-ICAT) approach for subcellular quantitative proteomics research.Methods1.Detection of mitochondrial alterations during apoptosis: The effects of HCPT on SMMC-7721 cells were measured by several methods including light microscopy, MTT assay, electron-microscopy, AnnexinⅤ-FITC and AO/EB staining; The apoptotic phase of mitochondria was ascertained by examination of changes in mitochondrial transmembrane potential, confocal microscopy and western blot analysis.2.Measurement of purity of isolated mitochondria: Mitochondria were isolated from hepatoma SMMC-7721 cells with mitochondria isolation kit and identified by Janus green B staining; Contaminations from cytosol, nucleus, and lysosomes were monitored by western blot.β-actin, Histone and Cathepsin-D protein were used as cytosol, nucleus, and lysosomes marker respectively; Contaminations from endoplasmic reticulum and cell membrane were monitored by detecting specific enzyme activity. Succinate dehydrogenase(SDH) was used as indicator of mitochondria purity, NADPH cytochrome c reductase(CR) and 5′-nucleotidase(5′-NT) were used as endoplasmic reticulum and cell membrane marker respectively.3.Analysis of differentialy expressed proteins in apoptotic mitochondria with 2DE-MALDI-TOF/MS: First, suitable conditions for two-dimensional electrophoresis were ascertained, including suitable loading quantity of sample, staining method and category of glue strip. Mitochondrial proteins were separated into three different fractions based upon their differing solubility. Mitochondrial proteins in three different fractions were separated by two dimensional electrophoresis respectively. Image analysis was performed using PDQuest(7.0) image analysis software. Matrix assisted laser desorption/isonization time of flying mass spectrometry (MALDI-TOF/MS) was adopted to identify differentia protein spots and then Mascot and PeptIdent software were used to search for proteins in MSDB and SWISS-PROT database. Bioinformatics analysis was performed on the differentialy expressed proteins. 4.Quantitative analysis of the mitochondrial proteome in HCPT-treated hepatoma cells using cleavable isotope-coded affinity tag(c-ICAT) strategy combined with two-dimensional liquid chromatography/tandem mass spectrometry(2D-LC/MS/MS): Mitochondrial proteins of intermediate solubility and mitochondrial hydrophobic proteins were identified and quantified with c-ICAT proteomics strategy respectively. The protein sample were labeled with c-ICAT reagent and digested with trypsin. The labeled cysteine-containing peptides were fractionated using strong cation exchange(SCX) and purified on an avidin column, sequentially the affinity tags were cleaved with reagent before the peptides were analyzed byμRP-HPLC-MS/MS. Quantitate proteins by integration labeled peptides elution peak areas, identify proteins from sequence information from MS/MS. Bioinformatics tools were used to analyze the proteins identified and quantified.5.Detection of purity of isolated nucleus and quantitative analysis of the nuclear proteome in HCPT-treated hepatoma cells: Nucleus were isolated from hepatoma SMMC-7721 cells with kit. Contaminations from cytosol, mitochondria, and lysosomes were monitored by western blot.β-actin, HSP60 and Cathepsin-D protein were used as cytosol, mitochondria, and lysosomes marker respectively. The nuclear proteins were labeled with c-ICAT reagent, digested with trypsin. The labeled peptides were fractionated using strong cation exchange and purified on an avidin column, then the tags were cleaved with reagent before the peptides were analyzed byμRP-HPLC-MS/MS. Quantitate proteins by integration labeled peptides elution peak areas, identify proteins from sequence information from MS/MS. Bioinformatics tools were used to analyze proteins identified and quantified.Results1. HCPT could remarkably inhibit the proliferation of SMMC-7721 cells and the IC50(50% inhibitory concentration) dose was about 80μg/ml. Some morphologic changes occured after cells were treated by HCPT, including phosphatidylserine(PS) was exposed from inner to outer leaflet of the plasma membrane, nucleus showed chromatin pyknosis and apoptosis, mitochondria was swollen, mitochondrial transmembrane potential was reduced and cytoehrome c released from mitochondria to cytosol.2. Stained with Janus green B, separated mitochondria showed some blue granule and strip structures. Western blot analysis showed that littleβ-actin, Histone or Cathepsin-D immunoreactivity had been detected in mitochondrial fraction. Detection of specific enzyme activity showed the specific enzyme activity of SDH in mitochondria was 13.8 fold compared with the value in homogenate. The specific enzyme activity of both CR and 5′-NT in mitochondria were little.3. The loading quantity of sample, staining methods and category of glue strips were selected as 200μg, silver staining and IPG Strips (3-10 NL) respectively via the optimizing conditions. Compared with control, thirty-nine mitochondrial protein spots showed differentia expression in HCPT-treated cells. Among them, 25 protein spots were up-regulated while 14 were down-regulated. Twenty differentia proteins were successfully identified by MALDI-TOF/MS.4. Ninety-one mitochondrial proteins of intermediate solubility were identified, among them, seventy-four proteins which were statistically significantly(P < 0.05) altered in HCPT-treated cells were quantified and identified. A total of forty-two proteins were significantly down-regulated, and thirty-two were up-regulated in response to apoptosis cells.5. 244 Mitochondrial hydrophobic proteins were identified statistically significantly(P < 0.05), a total of 154 proteins were quantified using shotgun proteomics method based on multiple dimensional liquid chromatography-linear ion trap /orbitrap mass spectrometer. Among them, compared with control cells, twelve proteins from apoptotic cells showed down-regulated, and 137 were up-regulated, five proteins showed no difference. In addition, thirteen alkali proteins, eleven proteins with Mr>200kDa and six proteins with Mr<10kDa were identified; fifty membrane proteins were identified. Moreover, there were forty-five proteins showed an elevation of more than 10-fold in apoptotic cells compared with control cells.6. Western blot analysis showed that littleβ-actin, HSP60 or Cathepsin-D immunoreactivity had been detected in nuclear fraction. Ninety-four nuclear proteins were analyzed statistically significantly(P < 0.05) using multiple dimensional liquid chromatography-linear ion trap /orbitrap mass spectrometer combined with c-ICAT strategy. A total of forty-three proteins were quantified, among them, twelve proteins showed down-regulated in HCPT-treated cells, thirty proteins showed up-regulated, and one showed no difference.Conclusions1. HCPT can induce SMMC-7721 cell apoptosis through the mitochondria apoptosis pathway.2. Mitochondria isolated with the methods mentioned above was pure enough to be used for the subsequent proteomic analysis.3. The twenty differentialy expressed mitochondrial proteins identified in this study might play very important roles in HCPT-mediated cell apoptosis.4. The function of the quantitatively differentialy expressed mitochondrial proteins of intermediate solubility were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton and so on.5. The hydrophobic proteins altered quantitatively were likely involved in life processes of cells including energy metabolism; cell structure; nucleic acid synthesis and metabolism; ribosome; proliferation, differentiation, apoptosis; and signal transduction. The results of this study will provide experimental foundation for further investigating the pharmacological action of HCPT at quantitative proteomics level, and prove the research strategies established in our study are helpful to the researches of subcellular proteomics and pharmic proteomics.6. In this part, the quantitatively differentialy expressed proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, nucleic acid synthesis and metabolism, structure of cell skeleton, energy metabolism. Several proteins were observed with a trend of up-regulation in mitochondria and nucleus after cells apoptosis, which is a meaningful clue to study the pharmacological action of HCPT and molecular mechanisms of apoptosis.更多还原