【作者】 金红；

【导师】 杨芃原；

【作者基本信息】 复旦大学 ， 分析化学， 2007， 博士

【摘要】 蛋白质组学作为当今分析化学与生命科学的学科交叉点,近年得到了迅速的发展。蛋白质组研究具有观察由多基因事件引起的多蛋白质组分整体变化的独特优势,比基因组学更接近生命现象的本质。它在医学和药物的靶向研究中具有强大的潜在价值,目前已被广泛用于研究各种疾病的发生、发展规律,并在寻找疾病的分子标志物的研究中取得了很多重大的进展。本论文主要包括以下几个部分:1.妊娠高血压综合征组织表达谱全谱分析;2.妊娠高血压综合征患者组织与正常组织的差异蛋白质组学研究:3.BeWo细胞在正常和缺氧条件下的差异蛋白质组分析;4.正常、胰腺癌组织和癌旁组织中差异表达蛋白的鉴定5.论文的相关研究:用RNAi干扰沉默14-3-3τ的研究以及鼠肝和法肝蛋白的RPLC/2-DE分离鉴定。论文取得的主要进展为:本论文从蛋白质组学层面深入对妊娠高血压综合征和胰腺癌两种常见的多发病的发病机制进行了基础研究,获得了迄今为止比较完善的蛋白质组信息;为病症的早期诊断和治疗探索了新的途径。论文初步建立和优化了胰腺癌组织的双向凝胶电泳技术,发现了与胰腺癌的发生相关的差异表达蛋白质,这些所发现的蛋白可能成为胰腺癌的蛋白标志物和药物治疗的靶蛋白。本文结果为对疾病标记分子的发现和细胞信号传递途径等方面的进一步研究提供了实验依据。论文进行了用RNAi干扰沉默14-3-3τ的研究;以及先以鼠肝为研究对象对RPLC/2-DE分离系统的可行性进行了验证,并运用到法国人肝样品的分离鉴定中,得到较好的实验效果,是对现有的研究方法和技术路线的有效的补充。一.论文首次建立了妊娠高血压综合征病人的蛋白质组表达谱,并运用研究方法中的双向凝胶电泳技术以及图像软件分析等蛋白质组学方法,比较了正常妊娠和子痫前期胎盘组织蛋白质组表达的差异;用胶内酶解、MALDI-TOF-TOF-MS以及数据库搜索对差异蛋白进行鉴定,并对其中特别重要的差异蛋白用Westernblot方法验证。另一方面,本论文采用激光显微切割技术切割组织并结合ICAT同位素定量联用LC-MS/MS技术,比较了正常妊娠和子痫前期胎盘组织蛋白质组表达的差异。结合这两种方法获得的数据,论文提出了对子痫前期发病的分子机制。1、妊娠高血压综合征组织表达谱全谱分析妊娠高血压综合征(简称妊高征)为常见的而又严重影响母婴安全的疾病,对其的防治是极为重要的。妊高征的病因至今未明,至今仍是根据其好发因素以及病理生理变化特点采取解痉、降压、利尿及适时终止妊娠等原则治疗,缺乏特效药物和预防疫苗。因此用蛋白质组学的理论和方法研究妊高征致病机理,寻找可用于诊断或治疗的靶蛋白有重要的意义。本论文建立了一个全面提取和分离妊高征胎盘组织总蛋白的技术方案,它包括激光显微切割妊高征胎盘组织、总蛋白的提取、一维的SDS-PAGE及色谱-质谱联用和两维shot-gun技术对总蛋白进行分离。然后对妊高征胎盘组织的总蛋白进行了表达谱的生物质谱鉴定,共鉴定了962个非冗余蛋白,其中包括泌乳刺激素等妊娠特异的蛋白、细胞骨架蛋白、热休克蛋白、与细胞周期,凋亡和信号转导等相关的蛋白,获得迄今为止最完整的妊高征胎盘组织的蛋白谱,为进一步研究妊高征的发病机理提供了丰富的实验数据;并为进一步分析检测低丰度蛋白奠定了基础。2、妊娠高血压综合征患者组织与正常组织的差异蛋白表达谱研究在建立了较完整的妊高症胎盘组织蛋白谱的基础上,本文继续应用两维凝胶电泳、两维高效液相分离等技术建立了妊娠高血压综合征和正常胎盘滋养细胞之间的蛋白质组图谱之间的差异谱。然后用胶内酶解、MALDI-TOF-TOF-MS以及数据库搜索对差异蛋白进行鉴定,鉴定了13个差异表达的蛋白,并对其中有重要意义的差异蛋白用Western blot方法进行验证,根据质谱鉴定结果,对蛋白进行初步分类和功能检索,在子痫前期胎盘中3个下调,10个上调,其中包括信号转导蛋白、分子伴侣、细胞骨架蛋白以及参与细胞内物质转运和能量代谢的酶类,这些蛋白都与子痫前期发病相关。另一平行路线为激光显微切割妊高征胎盘组织后,用ICAT同位素标记差异蛋白,用纳升级两维液相色谱.质谱联用技术鉴定蛋白,共鉴定了28个差异表达的蛋白,其中上调的有15个蛋白,下调的有13个蛋白;这些蛋白中有转铁蛋白、层纤维蛋白、胶原蛋白。对比分析了两种方法获得的差异蛋白,其中大部分蛋白都并不相同,这说明两种方法是互为补充的。3、BeWo细胞在正常和缺氧条件下的差异蛋白质组分析为了进一步研究妊高征发病的“胎盘滋养细胞缺氧”医学理论的分子机制,本文比较了缺氧培养和正常培养的滋养细胞之间蛋白质组图谱之间的表达的丰度差异;分析妊高征滋养细胞的蛋白质组表达谱和缺氧培养滋养细胞的蛋白质组表达谱之间的类似性和差异性。应用质谱分析技术鉴定那些可能是滋养细胞缺氧诱导妊高征机制有关的关键蛋白质组组分,确定是否为抑制蛋白质。应用了经典的分子生物学的方法验证这些蛋白质在妊高征发病中的作用。研究比较了缺氧条件和正常氧条件下,BeWo细胞分化成合体滋养细胞中蛋白质组表达的差异,并发现一些表达量有差异的蛋白。这些蛋白参与调控细胞氧化应激、细胞周期、以及信号传导,有助于进一步了解缺氧导致这些蛋白表达变化与子痫前期发病的关系。缺氧使BeWo细胞形成合体滋养细胞过程中,20个蛋白表达发生变化,包括抗氧化蛋白、参与糖酵解的酶类、分子伴侣、信号转导蛋白和细胞骨架蛋白等。20个蛋白中除了有个别蛋白质曾报道过与滋养细胞缺氧改变有关外,本文首次发现它们与缺氧影响合体滋养细胞形成有关。这些结果为研究滋养细胞缺氧反应性蛋白提供新的视角,并更好地了解缺氧影响合体滋养细胞形成的分子机制。二.正常、胰腺癌组织和癌旁组织中差异表达蛋白的鉴定论文的这部分工作建立和优化了胰腺组织提取蛋白的双向凝胶电泳技术,使之具有良好的分辨率和可重复性,应用双向凝胶电泳技术对12对胰腺癌组织和癌旁组织样品、3个胰腺良性疾病样品、3个正常胰腺组织样品进行分析,发现了30个差异表达蛋白质。用MALDI-TOF-MS/MS技术对差异表达蛋白质进行鉴定,共鉴定了24个蛋白质,其中15个蛋白质在胰腺癌组织中表达上调,9个蛋白质表达下调。有参与生命过程的各种酶及其前体,如胰蛋白酶前体、乙酰辅酶A脱氢酶等;有S100钙结合蛋白家族,如Calgizzarin(S100A11);有热休克蛋白家族:有细胞骨架蛋白家族;有与细胞膜转运有关的Annexin A5;还有与细胞组织有关的以及一些未知功能蛋白。这些蛋白质与胰腺癌的发生相关,可作为胰腺癌的蛋白标志物和药物治疗的靶蛋白。三、用RNAi干扰沉默14-3-3τ的研究及鼠肝和法肝的反相液相色谱结合双向凝胶电泳的分离路线本章工作用RNA干扰(RNA interference,RNAi)沉默14-3-3τ的表达,比较沉默前后的双向凝胶电泳图,找出差异蛋白,研究14-3-3τ在合体滋养细胞形成以及缺氧抑制合体滋养细胞形成中的作用。针对肝脏组织蛋白建立了反相高效液相色谱/双向凝胶电泳(RPLC/2-DE)的分离,初步建立了低丰度蛋白的鉴定模式,使该方法与双向凝胶电泳的方法互为补充,为高通量蛋白质组学研究提供了新的可能性。更多还原

【Abstract】 Nowadays, proteomics is an essential discipline as a current intersect of analytical chemistry and life science, proteomics develops rapidly in recent years. It observes all the components in whole proteomic level changes of the proteins caused by multi-genes and reveals the life essence more precisely than genomics. Proteomics has great potentials in medical and drug targets. Now proteomics is widely used as a tool for researching the cause and mechanism of various diseases, many great progresses have been made in finding disease biomarkers.Contents and advances of this dissertation described as follows.In the first chapter, advances in 2D and multidimensional separation techniques were reviewed in detail. Fundamentals of multidimensional separation techniques were introduced and the advantages and shortcomings of existing technical modes were discussed.Aims and advances of this dissertation were described.Chapter 2 described the total protein expression profile analysis of pregnancy induced hypertension (PIH) patients tissue. Chapter 3 described differential protein expression profile analysis between normal and PIH tissue. Chapter 4 described differential protein expression profile analysis between BeWo cell under normal and hypoxia conditions. Chapter 5 described differential protein identification between normal and pancreatic tumor tissue. And chapter 6 described some results of research, including fractionating complex protein samples from rat liver and French human liver prior to 2-DE using RPLC and differential protein expression profile analysis between normal and using RNAi technology to knock out 14-3-3τprotein.The main advances of this study can be summarized as follows. This study is focused on the fundamental of pathogenic mechanism of PIH and pancreatic cancer. A relative complete proteomic profile has been established, providing a new method for disease early diagnostic biomarker and treatment. 2D technology and the optimized experimental conditions for pancreatic cancer were established for this study use. The differential expressed proteins were found as the eventually disease biomarker and drug target for pancreatic cancer. Experimental findings of discovering disease biomarker and cell signal transduction pathways provide very useful information for the further study, In addition, RNAi to interfere and knock out 14-3-3τprotein to have functional validation have been studied.1. This is the first part of the dissertation. In this part, the proteomics study and resulted advances for PIH were described. A protein expression profile of PIH patient was established for the first time. By using proteomic technique of 2D-gel electrophoresis and image analysis software, the differential protein expression profile of tissue between the normal pregnant patient and PIH patient was compared. The differential proteins were identified via in-gel digestion followed MALDI-TOF-TOF-MS and database search; those especially essential proteins had been verified with Western blot. In addition, laser capture micro-dissection (LCM) technique was used to cut the sample tissue and then combined with hyphenation of ICAT with LC-MS/MS, for the comparision of the differential expressed protein profile between the normal pregnant patient and PIH patient. A molecular mechanism of PIH pathogenesis was proposed on the basis of the data resulted from both approaches.1.1 Total protein expression profile analysis of tissue of pregnancy induced hypertension (PIH) patientsPIH is a complex and serious condition of pregnancy, resulting in maternal and fetal complications even death if it is untreated or treated improperly. Up to now, there is no clinical measure to prevent this disease and treat it properly. Trophoblast cells in human placenta which can be separated from heterogeneous cells and collected by laser capture micro-dissection (LCM), plays an important role in early pregnancy. In this work, trophoblast cells in human placenta are considered as the functional protein in PIH. After total proteins have been extracted and identified by 1D-LC-MS/MS, 962 proteins of human trophoblast cell in PIH were analyzed by LTQ-Orbitrap. The identified proteins are classified according to molecular function, biological process and cellular component. Proteins of interest include14-3-3, which participate in signal transduction, heat shock protein 27 and annexinⅡandⅤin stress response like oxidation.A relatively complete protein data file of PIH was established, providing abundant experimental information for the further study of PIH pathogenesis mechanism, and laying a foundation of the further analysis of the low-abundance proteins.1.2 Differential protein expression profile analysis between normal and PIH tissueOn the basis of establishment of a relatively complete protein expression profile of PIH, a differential protein profile between PIH tissue and normal trophoblast cells was further established, via using 2D-gel, 2D-HPLC techniques for the separation of the whole extracted proteins. Afterwards, the differential proteins were identified via in-gel digestion combined with MALDI-TOF-TOF-MS, and then made a search with the database. 13 differential expressed proteins have been identified, especially essential proteins among these were verified with the Western blot for the validation of function. According to the MS identification and preliminary classification and functional search, it was found that 3 proteins in PIH were down-regulated and 10 proteins were up-regulated, they were signal transduction protein, molecular chaperon, cell skeleton protein and proteases which take part in material transportation and metabolism. All these proteins were related to the cause of preeclampsia.Another approach to the differential protein research was to cut the PIH tissue by LCM technique then mark the differential proteins by ICAT and identify them with 2D-LC-MS hyphenation technique. Total 28 differential proteins were identified. Among them, 15 proteins were up-regulated and13 were down-regulated. They are transferrin, laminin and collagen. It is noticeable that the proteins detected from the two different approaches are so different, meaning that two approaches should be complementary one another.1.3 Differential protein expression profile analysis of BeWo cells under normal conditions and hypoxia conditions Syncytiotrophoblast formation is affected by a number of pathological conditions and the suppressed syncytiotrophoblast formation due to hypoxia may play a role in the pathogenesis of preeclampsia. However, the molecular mechanism of hypoxia-inhibited trophoblast syncytialization is poorly understood. To determine the effect of hypoxia on trophoblast syncytialization, a proteomic analysis was performed in the human cytotrophoblast cell line BeWo using two-dimensional electrophoresis and MALDI-TOF-TOF-MS. Hypoxia induced marked inhibition of BeWo cell fusion and differentiation. The proteomic profiling was established under hypoxia in BeWo cell syncytialization. The results showed that twenty proteins were significantly up- or down-regulated under hypoxia, compared with cells under normoxia. In response to hypoxia, three antioxidants, peroxiredoxin 1, peroxiredoxin 2 and 1-Cys peroxiredoxin, were down-regulated, two proteins involved in glycolysis pathway (malate dehydrogenase and enolase) were up-regulated. The expression of two members of the annexin family (annexin A2 and annexin A5) was increased. We also found a decreased expression of 14-3-3τprotein in hypoxia treated cells. The expression of two cytoskeleton components (keratin 1 and b-actin) was found to be down-regulated. In addition, galectin-3 was up-regulated. These proteins have been implicated in regulating cellular oxidative stress, glycolysis, signal transduction, protein folding and degradation, cell mobility and cytoskeletal structure formation. Western blot analysis revealed that the levels of peroxiredoxin 1 and14-3-3τwere decreased, whereas the levels of annexin A5 and annexin A2 were increased in BeWo cells under hypoxia. These findings provided new insights into the molecular mechanisms in mediating cellular response to hypoxia in trophoblast syncytialization. 2. Differential protein identification between normal tissue and pancreatic tumor tissueThis part of work is mainly focused on the establishment of a method for pancreatic tissue total protein extraction and optimization of the working conditions for 2D gel, to achieve a high resolution and reproducibility. Twelve cancer samples and six normal tissue samples were analyzed. Total 30 differential expressed proteins were discovered. Among them, 24 proteins were identified by MALDI-TOF-TOF-MS, 15 proteins were up-regulated in pancreatic cancer and 9 proteins were down-regulated. Those proteins are various proteases and precursor proteins which take part in the life progress. They are trypsin precursor, coenzyme A dehydrogen protease; S100 calcium binding protein family like Calgizzarin; heat shock protein family; cell skeleton protein family; Annexin A5, correlated with membrane transportation; some other proteins related to cell and tissue and some unknown function protein. These proteins are related to the pancreatic cancer, and eventually become disease biomarker and drug targets.3. Differential protein expression profile analysis for the normal tissue and the RNA interference technology to knock out 14-3-3τprotein; fractionating complex protein samples from rat liver and French human liver prior to 2-DE using RPLCThis part of work is focused on RNA interference technology to knock out 14-3-3τprotein expression. Compared with before and after knocked out, differential expressed proteins were identified. The role of 14-3-3τprotein was validated in the formation of BeWo cells syncytialization under normoxia and hypoxia.An approach for fractionating complex protein samples from rat liver prior to 2-DE using RPLC was described. Experimental findings indicated that RPLC pre-fractionation provided an efficient approach to the low abundance proteins enrichment. This method is complementary to 2D-gel and can provide the new possibility for high throughput proteomic research. The proposed method has been applied to the the study of profiling mode of French Human Liver.更多还原