卵巢癌紫杉醇耐药细胞系的线粒体蛋白质组学研究

【作者】 贺豪杰；

【导师】 潘凌亚；

【作者基本信息】 中国协和医科大学 ， 妇科肿瘤， 2007， 博士

【摘要】 研究背景和目的:卵巢癌在妇科恶性肿瘤中发病率占第三位,而死亡率居第一位。5年生存率仅为15—20%。肿瘤细胞对化疗药物产生耐药性是造成卵巢癌5年生存率低的一个重要原因。已知的卵巢癌耐药机制包括:1)化学物质外流使肿瘤细胞内物质浓度降低。涉及的耐药基因和蛋白包括,MDR1,MRP和LRP等;2)肿瘤细胞对于抗肿瘤药物的转化和解毒功能增强,涉及谷胱甘肽S转移酶(GSTs)和P450家族等;3)化学药物作用靶分子的改变,如拓扑异构酶Ⅱ、二氢叶酸还原酶等;4)DNA损伤修复功能加强,如MGMT活性增强;5)化疗药物不能诱导肿瘤细胞凋亡,涉及的基因包括P53和Bcl—2家族等。从理论上来说,耐药相关基因在卵巢癌细胞中的表达应该能够预测肿瘤耐药和预后,然而目前多数研究表明耐药相关基因在卵巢癌的表达并不能良好地预测肿瘤的耐药和预后。肿瘤的发生需要细胞核和细胞浆的共同参与。细胞核基因组中的癌基因的表达被认为是肿瘤发生的主要原因,但有实验表明将非肿瘤细胞的细胞浆与肿瘤细胞融和能够使肿瘤的表现得到抑制,说明细胞核外存在着抑癌因素。线粒体是普遍存在于真核动物的一个重要的细胞器,是细胞进行氧化磷酸化,产生ATP的主要场所,内含有染色体以外唯一的DNA,并能进行转录和翻译;而且MtDNA可影响肿瘤细胞的抑瘤性。在上述卵巢癌的5种耐药机制中,至少有3种和线粒体相关:如化疗药物外流使肿瘤细胞内药物浓度降低的机制,肿瘤细胞对于抗肿瘤药物的转化和解毒功能增强的机制以及化疗药物不能诱导肿瘤细胞凋亡。所以选择线粒体为研究对象。蛋白质组学水平可以从蛋白质水平观察相应研究对象蛋白表达水平的变化,检测蛋白翻译后的修饰,同时找出与肿瘤耐药相关的蛋白质群,从而为寻找肿瘤标记物及肿瘤的早期诊断提供了极大的可能性。对于以蛋白质为主要成分的线粒体而言,采用蛋白质组学技术应该能够更加全面地分析和研究线粒体蛋白和卵巢癌耐药的相关性,同时为临床寻找克服肿瘤耐药性的靶点提供了新的途径。本研究在认识卵巢癌耐药相关基因的基础上,以卵巢上皮癌一线化疗药物紫杉醇耐药细胞系为研究对象,采用反复差速离心法分别提取紫杉醇敏感和耐药细胞系的线粒体,并用电镜和Western Blot法进行鉴定;提取线粒体蛋白,应用胍基化修饰的乙酰化稳定同位素标记、液相色谱—质谱联合制备紫杉醇敏感和耐药细胞系的线粒体蛋白质的表达谱和寻找差异表达蛋白。研究内容和方法:1.紫杉醇耐药细胞系SKOV3-TR、A2780TR耐药性的检测及化疗药物对线粒体膜电位的影响。对卵巢癌敏感细胞系SKOV3、A2780,以及其相应的紫杉醇耐药细胞系SKOV3-TR、A2780TR,培养并用CCK法检测耐药细胞系的耐药指数。运用JC-1染料结合流式细胞技术和荧光电镜技术对上述细胞系进行在不同紫杉醇浓度下线粒体膜电位检测,对不同细胞系的线粒体功能和对紫杉醇作用下线粒体功能的改变进行初探。2.优化细胞系线粒体蛋白质提取技术。分别采用试剂盒裂解法、经典的差速离心联合非连续密度梯度离心法和反复差速离心法分别提取紫杉醇敏感细胞SKOV3、A2780和其耐药细胞的线粒体,并用电镜和Western Blot法进行鉴定。3.卵巢癌细胞系SKOV3和A2780的线粒体蛋白质表达谱的制备和卵巢癌紫杉孵化疗敏感与耐药细胞系线粒体的比较蛋白质组分析。首次采用胍基化修饰的乙酰化稳定同位索标记法、液相色谱—傅立叶变换离子回旋共振质谱和定量分析软件MSAQ联用的定量蛋白质组策略制备卵巢癌细胞线粒体蛋白质表达谱,经过比较蛋白质学分析寻找差异表达蛋白。研究结果:1.本实验所用紫杉醇耐药的卵巢癌细胞系尚保持良好的耐药性,并且保持原有的细胞形态特点,耐药指数在SKOV3紫杉醇耐药细胞系和A2780紫杉醇耐药系分别为18.34±4.31和5.37±4.26。用荧光显微镜观察和流式细胞仪检测均可发现在紫杉醇为25μM时可造成线粒体膜电位的改变,但用流式细胞仪定量检测发现:在SKOV3紫杉醇敏感细胞系在药物浓度达到25μM时,线粒体膜电位有显著改变(P＜0.01),而在此浓度,耐药细胞系线粒体膜电位的改变尚未达到统计学显著性。2.本实验比较了裂解法、经典的差速离心联合非连续密度梯度离心法以及优化的反复差速离心法提取线粒体的效果,并且经电镜观察加以验证。从电镜结果可以看出,试剂盒裂解法线粒体无正常形态,破碎、断裂,仅剩双层膜碎片,线粒体纯度仅在40—50%。比较反复差速离心法和差速离心联合非连续密度梯度离心法,二者均能获得完整的线粒体,纯度可达到70%。比较二者线粒体蛋白的获得率,反复差速离心法用于SKOV3-TR及SKOV3细胞系,获取1mg线粒体蛋白质,需2×10~8个细胞;用于A2780-TR及A2780细胞系,获取1mg线粒体,约需3—4×10~8个细胞方。采用反复差速离心联合非连续密度梯度离心法,同样细胞量仅能提到约200—300ug的线粒体,且重复性较反复差速离心法差。因此,本实验最终采用反复差速离心法获得卵巢癌紫杉醇耐药和敏感细胞系的线粒体蛋白质,并以western blot方法对线粒体特异性蛋白抗体COX4、细胞核蛋白抗体Lamin-B、膜蛋白抗体Flotillin-1、胞浆骨架系统蛋白抗体β-actin等对线粒体的纯度进行鉴定,表明基本没有大量的胞浆、胞膜和胞核的污染。3.首次运用胍基化修饰的乙酰化稳定同位素标记法、液相色谱—傅立叶变换离子回旋共振质谱和定量分析软件MSAQ联用的定量蛋白质组策略,分别制备卵巢癌细胞系SKOV3和A2780的线粒体蛋白质表达谱。共鉴定出780个蛋白,两组细胞所共有的240个。鉴定蛋白的等电点(isoelectric point,PI)范围从4.09～11.36,PI＞10的蛋白有9个,＞8的蛋白达70个;分了量(molecular weight,Mw)范围从7.6kD至346kD。建立表达谱的同时,对两组细胞(SKOV3/SKOV3-TR、A2780/A2780-TR)进行比较蛋白质组学分析以寻找在两种不同的卵巢上皮癌细胞系中,耐药细胞株和敏感细胞株的差异表达蛋白。结果找出共同上调或下调的蛋白8个(＞2倍),有共同上调或下调趋势的蛋白28个(＞1.5倍)。其中线粒体蛋白Mimitin(myc induced mitochondriaprotein)为显著下调蛋白,是癌基因MYC作用产生的蛋白,有望成为耐药相关候选蛋白。结论:1.本实验所用紫杉醇耐药的卵巢癌细胞株尚保持良好的耐药性,并且保持原有的细胞形态特点。实验结果表明,紫杉醇可以引起线粒体膜电位的改变。卵巢癌细胞系体外经过紫杉醇作用诱导耐药后,引起其线粒体膜电位的改变的药物剂量应较化疗敏感的细胞株高,线粒体可能是耐药的靶点之一。2.反复差速离心法与差速离心联合非连续密度梯度离心法均可获得较高纯度的细胞系线粒体蛋白质。但前一种方法的线粒体获得率高,可重复性好。经westernblot正反相验证,基本没有大量胞膜、胞浆和胞核成分的混杂。3.运用胍基化修饰的乙酰化稳定同位素标记法联合液相色谱、质谱分析技术制备卵巢癌细胞系SKOV3和A2780的线粒体蛋白质表达谱获得成功。经过比较蛋白质组学分析表明,两种细胞细胞系有8个共同上调或下调(＞2倍)的蛋白质。其中,Mimitin在两组耐药细胞系均有差异显著的低表达,其可能成为耐药相关的标志候选蛋白。更多还原

【Abstract】 Background and objectiveOvarian cancer ranks the third in incidence of gynecologic malignancies and claims the highest mortality rate.Its five-year survival rate is only 15-20%.Chemotherapy resistance has been identified as one of the most important reasons for such low survival.Known mechanisms of chemotherapy resistance include:1) Chemotherapy drugs outflowing reduces the intracellular drugs concentration.Related genes and proteins include:MDR1,MRP and LRP et al.2) The antitoxic ability of carcinoma cells has improved.GSTs and P450 family are the related genes.3)The target that chemotherapy drugs work on has changed,such as topoisomeraseⅡ(TOPOⅡ),dihydrofolate reductase (DHFR).4) Repairing ability for DNA damage has been promoted.For example, MGMT activity has been enhanced.5) Chemotherapy failed to induce the apoptosis of cancer cells.P53 and Bcl-2 family are the related genes.Theoretically speaking,the expression of genes related to chemotherapy resistance in ovarian carcinoma cells could predict chemotherapy resistance or prognosis of the carcinoma.However,most reports failed to have the expected theoretic outcome.The reasons could be as follows:the tumor genesis needs the cooperation of both nucleus and cytoplasm,and expression of oncogenes of nuclear genome is regarded as the main reason. An interesting phenomenon has been found in several studies that emerging healthy cytoplasm and carcinoma nucleus could inhibit the development of carcinoma,indicating presence of anti-carcinoma products outside the nucleus.In eukaryotic cells,Mitochondria are critical subcellular organelles responsible for ATP generation through oxidative phosphorylation.It is the only place in addition to nuclei where DNA could be generated,transcripted and translated.Mitochondrial DNA(Mt DNA) can influence the anti-carcinoma ability of cells.At least three of the five chemoresistance mechanisms are related to mitochondria,such as drugs outflowing mechanism,improved antitoxic ability and apoptosis change of the carcinoma cells.Thus mitochondria have been selected as our study object.Proteomics approach provides a method for evaluating the expression of objective proteins,measuring the modified protein after translation and acquiring the chemoresistance related proteins concurrently,which offers a great opportunity to obtain a new marker of the malignancies and carry out early diagnosis of the disease.As the main component of mitochondria is protein,proteomics approach could comprehensively evaluate the association between mitochondria and ovarian chemoresistance and may acquire targets of the chemoresistance.Based on the identification of ovarian carcinoma chemoresistance genes,we have isolated and purified mitochondria from taxol sensitive and taxol resistant ovarian carcinoma cell lines by repeated fractions centrifugation and had them verified with electronic microscope and western blot method.Then,an Guanidination modified acetyl stable isotope labeling and LC-FTICR MS(liquid chromatography—hybrid linear ion trap fourier-transform ion cyclotron resonance mass spectrometry) strategy was performed to get the whole expression profiling of ovarian carcinoma mitochondria protein,as well as the different expression protein between chemotherapy sensitive and taxol resistant cells.Materials and methods:1.Measurement of chemoresistance of taxol resistant cell lines and evaluation of influence of taxol on MMP.Cultivated of the ovarian chemosensitive cell lines SKOV3,A2780 and corresponding chemoresistant cell lines SKOV3-TR,A2780TR and measured their resistance index with CCK-8 methods.After stimulating these cells with taxol of different concentration and staining them with JC-1 dyes,we examined the mitochondria membrane potential with fluoroscopy and Flow Cytometry to determine the influence of taxol on mitochondria function of different chemosensitive and chemoresistant cells.2.Optimized the methods of isolation of mitochondria from cell lines.Kit lysis method, classic fraction centrifugation combined with discontinuous density gradient centrifugation,and repeated fractions centrifugation were employed to isolate mitochondria from ovarian carcinoma taxol sensitive cell lines SKOV3,A2780 and their taxol resistant cell lines SKOV3-TR,A2780-TR,and mitochondria purity was verified by electron microscope and Western blot.3.Reportedly first use of Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.Result:1.The chemoresistant cells used in the experiment maintained excellent taxol resistant characteristics and the primary cell shape.The resistant indexes of SKOV3-TR and A2780-TR are 8.34±4.31 and 5.37±4.26,respectively.After being stimulated by 25uM taxol,the decreased mitochondrial membrane potential could be identified by fluorescent microscope and FCM.when the taxol concentration was 25uM,the taxol sensitive cell line SKOV3 exhibited significant change of MMP(P＜0.01).However, the taxol resistant cell line did not show a significant change at the same taxol concentration.It suggests that the mitochondrial functions of taxol sensitive cells and resistant cells influenced by taxol were different and mitochondria may be one of the important targets of taxol resistance.2.Comparing the methods of isolation of mitochondria,kit lysis method failed to show normal shape and only had cracked pieces with double membrane;the purity of mitochondria was about 40-50%.Much better isolation effects could be achieved by both optimized repeated fractions centrifugation and classic fractions centrifugation combined with discontinuous density gradient centrifugation.The purity of mitochondria could be up to about 70%.Next,efficiency of the two methods was evaluated.By repeated fractions centrifugation,1 mg mitochondria could be obtained from 2x10~8 SKOV3 and SKOV3-TR cells or 3-4x10~8 A2780 and A2780-TR cells.By fractions centrifugation combined with discontinuous density gradient centrifugation, only 200-300ug mitochondria could be isolated from the same amount of cells and the reproducibility of this method was worse.Therefore we selected repeated fractions centrifugation in our experiment and examined the isolated mitochondria with mitochondria marker COX4,nuclear marker Lamin-B,cell membrane marker Flotillin-1 and cytoskeleton proteinβ-actin by western blot method.It was shown that there were almost no contaminations of nuclei,cell membranes and cytoplasms.3.For the first time,Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy was used to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.About 780 proteins have been identified in total and 240 proteins of them are shared by the two groups.Eight proteins were significantly altered in both kinds oftaxol resistant cell lines(＞2 fold or＜0.5 fold).In addition,28 proteins showed trends toward significant alterations(1.5-2 fold or 0.67-0.5 fold).Among them,nuclei encoded mitochondrial protein Mimitin (Myc induced mitochondria protein) is the product of oncogene Myc transcripted, which could be one of the proteins associated with taxol resistance.Conclusion:1.The chemoresistant cell lines used in the experiment maintained excellent taxol resistant characteristics and the primary cell shape.The MMP of taxol sensitive cells and resistant cells influenced by taxol was different and mitochondria may be one of the important targets of taxol resistance.2.Both classic fractions centrifugation combined with discontinuous density gradient centrifugation and optimized repeated fractions centrifugation could produce mitochondria of high purity,however,the latter method had better efficiency and reproducibility.After being identified by western blot,there were no contamination of nuclei,cell membrane and cytoplasm.3.Guanidination modified acetyl stable isotope labeling and LC-FTICR MS strategy was performed to obtain the whole expression profiling of ovarian carcinoma mitochondria protein and the different expression protein between chemotherapy sensitive and taxol resistant cells.Mimitin expression decreased in both SKOV3-TR and A2780-TR cells and it could be one of the proteins associated with taxol resistance.更多还原