【作者】 戴志琴；

【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是死亡率最高的妇科恶性肿瘤。在肿瘤细胞减灭术的基础上辅助化疗的治疗策略虽然使疾病的预后得到改善,但晚期卵巢癌的5年生存率仍然徘徊在15-20%左右。肿瘤细胞对于化疗药物产生耐药性是主要原因之一。目前已知的卵巢癌耐药机制包括:1)化疗药物外流使肿瘤细胞内药物浓度降低。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)化疗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,如甲基鸟嘌呤甲基转移酶(MGMT)活性增加。5)化疗药物不能诱导肿瘤细胞凋亡。现有的研究结果表明,耐药基因在卵巢癌中的表达并不能良好地预测肿瘤的耐药性和预后,结合有关逆转肿瘤耐药性的研究在体内达不到理想效果,说明还有许多未知的耐药机制没有认识。线粒体是普遍存在于真核细胞中的一种重要的细胞器,是细胞进行氧化磷酸化,产生ATP的主要场所。细胞生命活动所需能量的90%是由线粒体提供的。与其它存在于细胞浆的细胞器相比,其特别引人注目之处在于,线粒体是唯一一个含有DNA(mtDNA)并能进行转录和翻译的细胞器。mtDNA具有遗传功能,其转录和翻译过程受到核基因的控制。近年的研究表明mtDNA在肿瘤发生、信号传导和凋亡调节方面亦具有重要作用。线粒体的化学组成主要是蛋白质,其含量占线粒体干重的65%—70%。由于mtDNA所含有的信息量小,线粒体所具有的一系列繁复的生物功能所需的90%以上的酶及自身结构蛋白主要依赖于核基因组与细胞质的蛋白质合成。在上述五种卵巢癌化疗耐药的机制中,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等,直接与肿瘤细胞的氧化磷酸化,能量产生的过程有关。抑癌基因p53所具有的诱导肿瘤细胞凋亡的作用更是直接通过线粒体的活动而执行这一功能。与耐药机制相关的一些酶本身就位于线粒体内。探讨线粒体基因组和蛋白质组在化疗耐药过程中的改变对于认识卵巢癌的耐药机制具有积极意义。受国家自然科学基金委员会资助,本课题组开展的线粒体基因组与卵巢癌耐药性及预后的研究,通过对初治和复发(耐药)的卵巢癌肿瘤标本各10例的mtDNA的全长测序,共发现69个基因库未记载的新的多态性位点和17个突变位点,突变率为0.54/10,000bp,初治与复发标本中mtDNA的突变率无明显的差别。由于临床标本受研究对象的个体差异、有无原发耐药性等因素的影响,其mtDNA的突变是否为肿瘤耐药特异性的改变仍不得而知,这些改变对于线粒体内蛋白质组表达的影响更无从答案。本研究在分别获得卵巢癌铂类敏感型细胞系SKOV3和A2780、及其顺铂和卡铂耐药型细胞株的基础上,以其作为体外化疗耐药的模型,全面探讨卵巢癌化疗耐药对于线粒体基因组和蛋白质组表达的影响;在不同的临床标本上进一步验证化疗敏感和耐药细胞系差异表达蛋白质的改变。研究内容本研究的内容和方法包括以下四个部分:1.分别提取SKOV3和A2780化疗敏感型和顺铂/卡铂耐药型细胞株的mtDNA,PCR扩增并全长测序。分析化疗敏感型和耐药型细胞株mtDNA碱基突变数目及相应的氨基酸变化,观察化疗耐药性对mtDNA改变的影响。2.优化细胞系线粒体蛋白质提取技术。分别采用经典的非连续密度梯度离心法与反复差速离心法,提取卵巢癌敏感型细胞株SKOV3和A2780、及其顺铂和卡铂耐药型细胞株的线粒体蛋白质,经过电镜观察法和Western Blotting法对线粒体的形态和纯度进行鉴定。3.采用差示凝胶电泳(Difference Gel Electrophoresis,DIGE)制备卵巢癌敏感和耐药型细胞株的线粒体蛋白质表达谱。经过胶上蛋白原位酶切和MALDI—TOF—MS质谱仪对差异蛋白点进行肽质量指纹谱分析。通过Mascot软件在SwissPort和NCBInr数据库进行检索,判断可能与耐药相关的差异候选蛋白。以Western Blotting法验证ATP-α等三种差异表达蛋白质。4.选择卵巢上皮癌化疗敏感和耐药患者的肿瘤标本各21例,应用免疫组化法验证耐药型A2780/CDDP和A2780/CBP共同下调的线粒体蛋白质ATP-α在卵巢上皮癌组织中的表达。研究结果1.与基因库中正常细胞的mtDNA序列相比对,化疗敏感型SKOV3与顺铂/卡铂耐药型细胞株SKOV3/DDP、SKOV3/CBP碱基位点的突变数目分别为31、34、38;导致氨基酸改变的数目分别为5、8、10。化疗敏感型A2780与顺铂/卡铂耐药型细胞株A2780/DDP、A2780/CBP碱基位点改变的分别为43、37、40;导致氨基酸的改变数均为9。该6株卵巢癌细胞株mtDNA全长序列的碱基位点突变率为22.4/10,000bp。比较铂类敏感型与耐药型细胞株之间mtDNA碱基位点的突变情况,母代SKOV3(铂类敏感型)细胞经过体外铂类药物诱导耐药后,SKOV3/CDDP mtDNA的55个碱基位点较母本发生了突变;SKOV3/CBP中52个碱基位点较母本发生了突变。母代A2780经过体外铂类药物诱导耐药后,A2780/CDDP mtDNA的17个碱基位点较母本发生突变;A2780/CBP中12个碱基位点较母本发生突变。2.采用经典的差速离心/非连续密度梯度离心和反复差速离心两种方法提取卵巢癌细胞系线粒体蛋白质均获成功。以Western Blotting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等进行验证,结果显示两种方法提取的线粒体纯度均较好。电镜观察线粒体的纯度和形态,前一种方法所得的纯度虽高,但线粒体碎片较多;而后者所得的线粒体纯度好、线粒体碎片较少。比较线粒体的得率,收集1*10~8个细胞,前者获得的蛋白量约50ug(±);后者约250ug(±)。本研究选择了反复差速离心法作为最终提取线粒体蛋白质的方法。3.通过差示凝胶电泳(Difference Gel Electrophoresis,DIGE)与MALDI—TOF—MS质谱鉴定相结合的方法,分别对卵巢癌化疗敏感与耐顺铂和卡铂耐药细胞株,SKOV3与SKOV3/DDP、SKOV3与SKOV3/CBP、A2780与A2780/DDP、A2780与A2780/CBP进行比较蛋白质组分析,共显示出236个差异表达点,其中表达上调点为108个,表达下调点为128个。以差异的倍数高或两个以上细胞株共同具备的差异表达点为依据,对其中的19个蛋白差异点进行鉴定。结果表明,共鉴定出13个蛋白点。除3个为未明确蛋白外,其余10个均为下调蛋白,其中的5个为线粒体功能相关蛋白,分别为ATP-α、PRDX3、PHB、ETF、ALDH,均为核编码蛋白,多与线粒体的有氧能量代谢和呼吸链电子传递有关。在这5个差异蛋白质中,仅有ATP-α在A2780/CDDP和A2780/CBP耐药型细胞株中共下调。购得ATP-α、PRDX3、PHB三种抗体,采用Western Blotting法验证表明,三种蛋白质分别在耐药细胞株A2780/CDDP和A2780/CBP、SKOV3/CBP中表达下调。4.应用免疫组化法验证耐药型A2780/CDDP和A2780/CBP共同下调的线粒体蛋白质ATP-α在卵巢上皮癌组织中的表达。结果表明,化疗敏感组和耐药组各21例,两组患者的年龄、分期和组织学分类无差异。从免疫组化结果的数值看,化疗敏感组中ATP-α的表达率高于化疗耐药组,但两组不具备统计学差异(p=0.25)。研究结论1.卵巢癌化疗敏感和耐药细胞株mtDNA全长序列的碱基位点的突变数目为31～41个,高于本课题组关于卵巢癌组织中mtDNA的碱基位点突变数(17个)。卵巢癌细胞系体外经过铂类药物诱导耐药后,mtDNA较母本发生较明显的突变。但是,化疗药物作用后mtDNA的突变数SKOV3较A2780明显增多。2.采用经典的差速离心/非连续密度梯度离心和反复差速离心法提取卵巢癌细胞株线粒体均能获得高纯度的线粒体蛋白质。反复差速离心法用于细胞株线粒体蛋白质的提取具有线粒体得率较高,线粒体碎片相对较少的优点。3.差示凝胶电泳(DIGE)在传统双向电泳(2-D)技术的基础上,结合了多重荧光分析的方法,完成了本研究中线粒体蛋白质样本量较低的比较蛋白质组学分析。其所具备的独特的内标概念,使计算机软件系统全自动地根据每个蛋白点的内标对其表达量进行校准,从而保证了结果的准确性、可靠性和重复性。本研究中所鉴定出的部分细胞差异表达蛋白ATP-α、PRDX3、PHB、ETF、ALDH等均为核基因编码的蛋白质,均表达下调的蛋白,多与细胞的能量代谢和呼吸链电子传递有关。研究中未观察到线粒体mtDNA所编码的蛋白质改变可能与mtDNA所具有的遗传信息有限以及实验技术的局限性有关。4.应用免疫组化法验证线粒体蛋白ATP-α在卵巢上皮性癌组织中的表达,观察到ATP-α在化疗耐药组患者的卵巢癌组织中表达下调,但是在统计学上未达到显著性意义。更多还原

【Abstract】 Background and objective:The mortality of ovarian cancer is the highest among the gynecological malignant.The prognosis of ovarian carcinoma has been improved by chemotherapy based on cytoreductive(CRS),however,5 years survival rate of the advanced disease is still between 15-20%,the drug resistence of tumor cells is the one of the main causes.The known mechanisms of chemo-resistance of ovarian cancer included:1) Outflow of chemotherapy drug decreases concentration in cells.MDR1,MRP和LRP are the mostly involved resistant gene and protein;2) Conversion and detoxification of anti-tumor drug by carcinoma cells is enhanced,involving GSTs and P450;3) Chemotherapy drugs result in modification of target molecule on cells,including TOPⅡand reductase of dihydrofolic acid;4) Enhancement of DNA repair,including MGMT;5) Chemotherapy drug was unable to induce apoptosis of cancer cells.The present studies have revealed that expression of drug resistance genes of OC fails to predict the drug resistance and prognosis,the study of retroconversion of drug resistance fail to be an ideal approach, which indicates that there remains much unknown the mechanism of drug resistance.Mitochondria,which is a vital cell organella universally in eukaryocyte,,provides sites for oxidative phosphorylation and generation of ATP,and is responsible for 90%of the energy for celluler metabolism.Differing from other organella,it is distinctive for containing DNA and ability to transcription and translation.mtDNA can inherit,the transcription and translation of which is under the regulation of nuclear gene.Recent researches have indicated that mtDNA plays an important role in tumorigenesis,signal transmission and apoptosis.Mitochondria mainly composed of protien which accounts for 65%—70%dry weight of the total.As mtDNA contains limited information,the enzymes and structure protien underlying the 90%of cellular function depend on nuclear gene-coded.Oxidative phosphorylation and energy production in carcinoma cells are directly related to part of the mechanisms of drug resistance mentioned above,including(namely) drug decreased drug concentration in cells for outflow of chemotherapy and enhancement of conversion and detoxification of anti-tumor drugs.Moreover,Mt are involed in the apoptosis of tumor cells induced by p53.In addition,some of the drug resistance related enzyme are just located in mitochondria.Therefore,mitochondria deserve investigation concerning genome and proteome as it is associated with drug resistance.Funded by national natural science committee,our team has explored in the field of mitochondrial genome and its relation to drug resistance and prognosis of OC.10 specimens of mtDNA sequence of primary OC and recurrent(resistant) OC have been conducted and new polymorphism and 17 mutations(mutational rate 0.54/10,000bp) were identified.There is no significant difference between the mutational rate of mtDNA in initial treatment specimen and that in recurrent specimen.Considering individual variation among subjects and possible influence of primary resistance,it remains to be elucidated whether the mtDNA mutation is specifically related to drug resistance as well as the effects on expression of proteome are caused by these mutations.In the study,the cisplatin resistance cell lines and carboplatin resistance cell lines derived from platinum sensitivity SKOV3 and A2780 served as the models of drug resistance induced by chemotherapy in vitro.We studied the effects of chemotherapy resistance on mtDNA mutation and expression of proteome.We further verified different expression protein in drug-sensitive and resistant specimens under different clinical conditions.Material and Motheds1.Extract the mtDNA from SKOV3 line,A2780 line,cisplatin resistant cell lines and carboplatin resistant cell lines,and then amplified them by PCR for sequencing.Analyse the sequences of the chemotherapy sensitive cell lines and the chemotherapy resistant cell lines as well as their corresponding alterations of amino acids.Observe the effects of chemotherapy resistance on mtDNA mutation.2.Optimized isolation technology for mitochondria from cell lines:classical discontinuous density gradient centrifugation and repeatedly fractions centrifugation were introduced for isoltion of mitochondria from SKOV3,A2780,and their respeative cisplatin resistant lines and carboplatin resistant lines.Observation of the morphology and evaluation of purity was performed with electron microscopy and Western Blotting.3.DIGE was used for presenting protein expression spectrum of mitochondria from chemotherapy sensitive cell lines and chemotherapy resistant cell lines.Using sequence-specific proteases to break up the proteins into peptides on the gel was performed and A peptide-mass map was generated to detect different expression proteins by MALDI—TOF—MS (mass spectrometer).Search for possible candidate chemo-resistance associated protein in SwissPort and NCBInr databases.Verified the three differential expression proteins with Western Blotting.4.The expression of mitochondrial protein ATP-α,which had been down-regulated in the drug-resisted cell lines A2780/CDDP and A2780/CBP,was reevaluated in 21 chemotherapy-sensitive OC specimens and 21 drug-resistant OC specimens with immunohistochemistry.Results:1.Compared with the normal cells,31、34 and 38 mutations occurred respectively in the mtDNA of SKOV3,SKOV3/DDP and SKOV3/CBP lines,with5,8 and 10 corresponding amino acids changes,respectively.43,37 and 40 mutations occurred respectively in the mtDNA of A2780,A2780/ DDP and A2780/ CBP lines,with 9 corresponding amino acids changes respectively均为9。For the 6 cell lines,the mutation rate of the mtDNA was 22.4/10,000bp. After induction of drug resistance in vitro,SKOV3/CDDP presented 55 mutations in mtDNA compared with their mother generations(SKOV3);SKOV3/CBP presented 52(compared with SKOV3);A2780/CDDP presented 17(compared with A2780);and A2780/CBP presented 12 mutations(compared with A2780).2.Successful isolation of mitochondria from OC cell lines was obtained by both classical uncontinuous density gradient centrifugation and repeatedly fractions centrifugation. Western Blotting revealed satisfactory purity was revealed by western blot.Under the electron microscopy,high purity with more mitochondrial debris was observed with density gradient centrifugation while high purity with less mitochondrial debris with differential centrifugation.As more protein was extracted by fractions centrifugation,finally,this technology was adopted in our study.3.Combination of DIGE and MALDI—TOF—MS was introduced for compare-proteome analysis of the following lines:SKOV3 and SKOV3/DDP、SKOV3 and SKOV3/ CBP、A2780 and A2780/ DDP,A2780 and A2780/ CBP,and 236 differential expression spots were discovered including 108 up-regulation and 128 down-regulation. Nineteen differential expression spots,whose expression differences significant or presented in more than 2 lines,were selected for identification.13 protein spots were identified and 10 were down regulated,5 of which were mitochondrial protein,namely,ATP-α、PRDX3、PHB、ETF、ALDH,coded by nuclear DNA and responsible for oxidative metabolism and electron transport respiratory chain.Of the 5 proteins,ATP-αis the only one down regulated in two lines,A2780/CDDP and A2780/CBP,down regulation of PRDX3 and PHB in SKOV3/CBP has been verified by Western Blotting as well.4.ATP-αin ovarian cancer tissue was verified by Immunohistochemistry.No statistical difference(p=0.25) was found between the chemotherapy sensitive group and chemotherapy resistant group,despite that a higher ATP-αexpression was observed in the chemotherapy sensitive group.Conclusions:1.There were 31～41 mutations in mtDNA from sensitive ovarian cancer cell lines and resistant lines,more than those from ovarian cancer tissue.There were much more mutations of mtDNA of drug-resistant SKOV3 than drug-resisted A2780.2.High purity mitochondria were obtained from ovarian cancer cell lines by classical uncontinuous density gradient centrifugation and repeatedly frantions centrifugation. repeatedly fractions centrifugation is superior due to higher extraction with less mitochondrial debris.3.DIGE,characterized by integrity of 2-DE technique and CyDye DIGE fluors, successfully processed the comparative proteomic analysis with lower amount of mitochondrial protein.With a specific internal standard integration,the software automatically directs the rectification of expression of protein.Thus the results are more accurate,reliable and reproducible.We identified some differentl expression proteins,such as ATP-α、PRDX3、PHB、ETF、ALDH,which are coded by nuclear genes and all down regulated.These proteins are associated with energy metabolism and electron transfer respiratory chain.No proteins encoded by mtDNA was observed in the different expression proteins and it may be related to limited genetic informations in mtDNA and limitation by experimental techniques.4.Immunohistochemistry to detect ATP-αexpression in ovarian cancer tissue demonstrated a down -regulation of ATP-αin ovarian cancer tissue of patients with drug-resistance,however,without statistical significant differences.更多还原