【作者】 孙成荣；

【导师】 唐建武；

【作者基本信息】 大连医科大学 ， 病理与病理生理学， 2007， 博士

【摘要】 背景:浸润性生长和转移潜能是恶性肿瘤不同于良性肿瘤的根本特征,是恶性肿瘤顽固性和难治性的根本原因。由转移所导致的恶性肿瘤患者死亡率高、预后差及其发生机制不清,长期以来一直是肿瘤学研究面临的难题之一。由上皮来源的恶性肿瘤,淋巴道转移是其早期转移的主要方式,因此对于淋巴道转移机制进行研究,发现淋巴道转移关键分子机制和通路,并建立起一套行之有效的针对性阻遏手段,将对恶性肿瘤患者具有重要的现实意义.。Hca-F和Hca-P是一对来源于同一亲本细胞、且淋巴道转移潜能显著不同的小鼠肝癌腹水型细胞株,其中Hca-F为高淋巴道转移力细胞株(> 70%),Hca-P为低淋巴道转移力细胞株(<30%),二者在肿瘤淋巴道转移机制研究中互为参照,是难得而有效的细胞模型。我们已应用基因芯片等高通量技术手段,在mRNA水平对Hca-F细胞株和Hca-P细胞株进行研究,发现二者有明显差异的基因表达。但是从mRNA水平进行研究,只包括了转录水平的调控,不但在丰度上不能反应蛋白质的表达水平,而且蛋白质复杂的翻译后修饰、亚细胞定位和迁移、蛋白质和蛋白质的相互作用等,也几乎无法从mRNA水平来判断。蛋白质是生理功能的执行者,是生命现象的直接体现者,对蛋白质的研究将直接阐明生命在生理和病理条件下的变化机制。蛋白质组学是对一个细胞或组织的基因组所包含的全部蛋白质进行的研究。但由于技术方法限制及蛋白质表达的时空差异,很难分离和得到表达的所有蛋白。因此发展可靠高通量的技术手段,将会推进对蛋白质组的研究。为实现这一目标,以双向凝胶电泳和质谱为基础的蛋白质组学分离和鉴定技术迅速发展起来。目的:本研究应用近年来发展起来的定量蛋白质组学技术—荧光差异双向凝胶电泳,在蛋白质水平对高、低淋巴道转移力小鼠肝癌腹水型细胞株Hca-F和Hca-P基因表达进行比较研究,建立相应的荧光差异双向凝胶电泳蛋白表达图谱,并对有统计学意义的差异性蛋白质点进行质谱分析,以筛选肝癌淋巴道转移相关蛋白,为进一步阐明淋巴道转移的分子机制奠定基础。方法:将冻存的Hca-F与Hca-P细胞株复苏后,进行细胞培养。以动物实验观察二者在615小鼠的淋巴结转移率。提取总蛋白的瘤细胞洗涤后,冷冻离心机离心,取沉淀加入细胞裂解液,并在冰浴下超声破碎。冷冻离心机离心后,提取上清。采用蛋白定量标准试剂盒,测定样品蛋白浓度。蛋白的荧光标记按照厂家介绍的步骤进行,Cy3(Cy5)染料分别交叉标记Hca-F(Hca-P)细胞提取的蛋白,Cy2染料标记总量相同,但Hca-F细胞和Hca-P细胞各1/2量蛋白。与缓冲液及IPG泡涨液混合,制成一向等电聚焦体系。在该体系中重泡涨IPG干胶条10个小时后,按照操作说明使用IPGphⅡ电泳仪,以50mA电流等电聚焦76KVh。等电聚焦结束的IPG胶条平衡后,进行12.5% SDS-PAGE电泳。双向凝胶电泳结束后,应用TyphoonTM9400成像系统扫描成像。采用DeCyder软件DIA和BVA模式,进行凝胶图像分析。不同凝胶内分析模式采用配对比较,以Cy2为内标计算比值,通过Cy3 /Cy2及Cy5 /Cy2的比率对应每个蛋白点含量,并对其进行标准化计算。重复4块胶用来计算每个蛋白点平均量的改变,并进行t检验,计算p值,设定p<0.05为有显著性差异。经DeCyder软件分析有统计学意义的蛋白质点,经自动蛋白质点切割机切取后,转移至96孔板中,进行胰蛋白酶胶内酶解,然后用于电喷雾电离串联质谱进行分析,通过Sequest软件检索NCBInr数据库鉴定蛋白质。以蛋白质印迹验证差异蛋白质Erp29和UCHL3的表达水平。对鉴定出的差异性蛋白质进行生物信息学的功能分析,所使用的分析系统为DAVID和PANTHER,分别从蛋白质表达的细胞内位置、分子功能、生物学过程以及代谢和信号转导通路上进行分析。结果:Hca-F细胞株和Hca-P细胞株的成瘤率100%,淋巴结转移率分别为75%和25%,两者相比的统计学检验有显著性意义(p<0.05=,符合本实验对细胞模型的要求。建立Hca-P和Hca-F细胞株的蛋白质荧光差异双向凝胶电泳蛋白表达图谱。经分析,Hca-F和Hca-P样品间有统计学意义的差异性蛋白质点共163个,其中比值改变2倍以上的差异性点23个;1.5倍~2倍的差异性点79个;1倍~1.5倍差异性点61个。在Hca-F细胞中上调的蛋白质点为86个,下调的蛋白质点为77个。将这些差异性蛋白质点进行质谱分析,获得MS/MS串联质谱图后应用Sequest软件在NCBInr数据库中检索,共得到168种蛋白质,其中2倍以上差异性蛋白质有缬酪肽蛋白(Vcp)、真核翻译延长因子2(Eef2)、转羟乙醛酶(Tkt)、M2型丙酮酸激酶(Pkm2)、预测的谷氨酰胺酶异构体5(Gls)、Ero1样蛋白(Ero1)、Aldh2蛋白(Aldh2)、假定蛋白LOC192169(1810047C23Rik)、RuvB样蛋白、(Ruvbl1)、真核翻译延长因子1α2(Eef1α2)、波形蛋白(Vim)、外周蛋白(Prph)、结蛋白(Des)、膜联蛋白7(Anxa7)、脑肌酸激酶(Ckb)、线粒体苹果酸脱氢酶2(Mdh2)、膜联蛋白5(Anxa5)、过氧化物酶体的烯酰辅酶A水解酶1(Ech1)、不均一核糖核酸A2/BI异构体1(Hnrpa2b1)、内酰胺酶β2(Lactb2)、谷胱甘肽S-转移酶ο1(GSTο1)、泛素C末端水解酶同工酶L3(UCHL3)、内质网蛋白29前体(ERp29)、溶血磷脂酶1(Lypla1)、Crk蛋白(Crk)、微管不稳定蛋白(Stmn1)。蛋白质印迹验证差异性蛋白,其结果表明Erp29和UCHL3在Hca-P细胞中均高表达,与鉴定结果一致。对全部168个差异性蛋白进行生物信息学分析,在细胞成分分析中,差异性蛋白质多位于细胞器,其中以线粒体、细胞骨架、内质网居多;分子功能分析显示,差异性蛋白质涉及分子伴侣、细胞骨架蛋白、水解酶、裂解酶、转运蛋白、核苷酸结合蛋白、氧化还原酶、蛋白酶、选择性钙离子结合、选择性调节分子、转录因子、转移酶等功能,其中具有核苷酸结合蛋白分子功能的蛋白质最多占19.4%,其次为氧化还原酶及细胞骨架蛋白各占12.1%;生物过程分析显示这些差异性蛋白参与到各种生物学过程中,其中主要参与到蛋白质代谢和修饰的蛋白质占29.1%,核酸及核苷酸代谢的蛋白质占16.4%,细胞结构和移动的蛋白质占12.7%,免疫和防御的蛋白质占12.1%;代谢和信号转导通路的分析显示上述差异性蛋白涉及精氨酸与脯氨酸代谢、糖酵解和糖异生、碳固定、细胞交流、肌动蛋白骨架调节、黏着斑、抗原的加工和提呈等代谢和信号转导通路。结论:以高低淋巴道转移力小鼠肝癌细胞株Hca-F和Hca-P为研究对象,采用定量蛋白质组学技术—荧光差异双向凝胶电泳,建立了高低淋巴道转移力小鼠肝癌细胞荧光差异蛋白表达图谱,高通量筛选与肿瘤淋巴道转移相关的蛋白。共得到163个有统计学差异的蛋白质点,在高淋巴道转移力细胞株Hca-F中表达上调的蛋白质点为86个,在低淋巴道转移力细胞株Hca-P中表达上调的蛋白质点为77个。以质谱技术分析及数据库搜索共鉴定出168种蛋白质,对这些蛋白质进行生物信息学的分析,分别从这些蛋白质表达的细胞内位置、分子功能、生物学过程以及代谢和信号转导途径上进行分析。分析结果显示差异性蛋白质多位于细胞器中,以线粒体居多;具有核苷酸结合蛋白分子功能的蛋白质最多,其次为氧化还原酶与细胞骨架蛋白;蛋白质代谢和修饰、核酸及核苷酸代谢、免疫和防御等生物学过程在肿瘤淋巴道转移中发挥了较为重要的作用;黏着斑、肌动蛋白骨架调节及抗原的加工和提呈等信号转导通路可能是肿瘤淋巴道转移中的关键性途径。更多还原

【Abstract】 Background: Invision and metastasis are the fundamental characters in differentiating the malignant tumer from benign one,and are the major causes of refractoriness of tumer . High mortality ,poor prognosis and uncertain pathogenesis deriving from metastasis of malignant tumor are still one of tough problems in tumor research for prolonged time.Because majority of malignant tumors are carcinomas and lymph node metastases often represent the first step in the metastatic process,it is very important to make the molecular mechanisms of lymphatic metastasis clearly,and to construct a set of efficient repression methods.Hca-F and Hca-P are a pair of syngenetic mouse hepatocarcinoma ascites cell lines ,presenting a specific potential of lymphogenetic metastasis when inoculated subcutaneo- usly in 615 mice. Hca-F with a highly metastastic potential (>70%)and Hca-P with a low one( < 30%)are rare and available cell model as a reference each other in the research of the molecular mechanisms of lymphatic metastasis.We have investigated the gene expression of Hca-F and Hca-P in mRNA level using cDNA microarray,and have found the conspicuous differential gene expression. However,studying in mRNA level only represents the transcriptive regulation,and can’t reflect the abundance ,the complicated post-translation modification,location and transition in subcellar and the interaction of proteins etc.Protein is a executant of physiological function and a direct performer of life.The mechanisms of physiology and pathology will be elucidated by investigating protein.Proteom is to study all the proteins in a cell or in a tissue.It is difficult to sepratate and to abtain all the expressed proteinsowing to the restriction of the modern technique.So developing high-through technical methods will facilitate proteomics.To get the destination, two-dimensional gel electrophoresis(2DE) and mass spetromery (MS)as the basic techniques of sepratation and identification have been developed.Object: In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F (highly metastatic)and Hca-P(low metastatic) were compared using fluorescent two-dimensional difference gel electrophor- esis(2D DIGE)-quantitative proteomics which has been developed recently,and then differential protein spots with statistical significant between Hca-F and Hca-P were identified by mass spetromery (MS).Method: The anabiosis cells of Hca-F and Hca-P were intraperiton- eally injected to culture 2 passage in 615 mice,and cultured in vitro for 24 hours.Collected tumor cell,which of half part for the animal experiment to detect the metastaic rate of lymphtic nodes,another half one for extracting total protein. Hca-F and Hca-P were injected subcutaneously in the hypoderma of right axillary line to observe 28 days ,and executed 20 mice. All draining lymphtic nodes were exsected to be stained with HE . The metastaic rate of lymphtic nodes were calculated.The tumor cells were washed and were centrifuged in a refrigerated centrifuge. The deposition was transfered into a reaction tube containing lysis buffer Lysates to be sonificated,and were centrifuged in a refrigerated centrifuge. Supernatant was total protein,and protein concentration of lysates were determined by Bradford assays. Labeling reactions using CyDyes were performed following the manufacturer’s instructions. For the preparation of the“internal pooled standard,”equal protein amounts of the two corresponding twins were pooled and labeled with Cy2. Equal protein amounts of Cy2, Cy3, andCy5 labeled samples were mixed and added to an equal volume sample buffer and rehydration buffer .DryStrips were rehydrated for 10 hours in rehydration buffer. IEF was performed using IPGphⅡ.After anodic cup loading, the IPG strips were focused for a total of 76 kVh at 50mA. Prior to electrophoresis each strip was equilibrated in equilibration buffer . 2-DE was performed using a stacking gel and a separation gel. The gels werescanned with a Typhoon 9400 using the parameters suggested by the manufacturer.Image analysis was performed with DeCyder. The differential protein spots with statistical significant were excised by an automated spot excitation robot, recovered the spots in a 96-well plate. Protein identification was performed by an in-gel digestion method. Mass spectrometric analysis of tryptic digests was performed usingLC- ESl- MS/MS. Sequest software researched NCBInr database to identify proteins. The identified proteins were verified by Western blotting.All the identified proteins were analized by DAVID and PANTHER at cell component, molecular function ,biological process and metabolism and pathway.Result: The rates of tumor formation of Hca-F and Hca-P cells were both 100%,and the metastatic rates of lymphtic nodes were 75% and 25% respectively,with statistical significance(p<0.05). Hca-F and Hca-P cell lines consistented with the requirement of the experiment.The differential expression of protein profile of Hca-F and Hca-P have been established.DIA mode of DeCyder may detect three overlapping images in one gel ,labeled with Cy2, Cy3 and Cy5 respectively ,and may abtain precise radio. BVA mode of DeCyder may match among the gels ,and may detect the diference which can be calculated level of confidence by statistic in all the gels . 163 differential protein spots with statistical significance between Hca-F and Hca-P were detected, including 23 differential spots of more than 2 fold; 79 of 1.5 fold to 2 fold; 61 of 1 fold to 1.5 fold. 86 protein sprots were up-regulated in Hca-F and 77 protein spots were down-regulated in Hca-F. All of the protein differential spots were digested and analyzed using MS,and 168 proteins were identified by NCBInr database using sequest software.The differential expression proteins more than 2 fold : Vcp, Eef2, Tkt, Pkm2 ,Gls,Ero1, Aldh2, 1810047C23Rik, Ruvbl1, Eef1α2, Vim, Prph, Des, Anxa7, Ckb, Mdh2, Anxa5, Ech1, Hnrpa2b1, Lactb2, GSTο1, UCHL3, ERp29,Crk, Lypla1, Stmn1. The identified proteins were verified by Western blotting. ERp29 and UCHL3 were down-regulated in Hca-F ,as same as the result of identification.The analysis of bioinformatics of 168 proteins revealed that the identified proteins were principally located in intracellular organelle,incluing mitochondrion, cytoskeleton, and endoplas- mic reticulum etc.The identified proteins are involved chaperone ,cytoskeletal protein , hydrolase , nucleic acid binding , oxidoreductase , protease , select calcium binding protein , select regulatory molecule , transcription factor , transfer/carrier protein ,transferase etc.The analysis of biology process revealed that the identified proteins mainly participate in protein metabolism and modification, nucleoside, nucleotide and nucleic acid metabolism, cell structure and motility, immunity and defense etc. The analysis of metabolism and pathway revealed that the identified proteins mainly participat in arginine and praline metabolism,proteasome, glycolysis/gluconeogenesis,carbon fixation,cell communication,regulation of actin cytoskeleton,focal adhension, antigen processing and presentation etc.Conclusions: In order to obtain lymphatic metastasis-associated proteins, the protein expressed profiles of mouse hepatocarcinoma ascites syngeneic cell lines Hca-F (highly metastatic)and Hca-P(low metastatic) were compared using 2D DIGE,followed by MS to identify the differential protein spots with statistical significant. High-through quantitative proteomics technique is a powerful tool to identify lymphtic metastasis- associated proteins. 163 differential protein spots in 2D DIGE were detected, including 86 protein spots up-regulated in Hca-F and 77 protein spots down-regulated in Hca-F. Followed by MS and researching in database,168 proteins were identified. The analysis of bioinformatics of the identified proteins revealed that they were principally located in intracellular organelle,especially in mitochondrion; nucleic acid binding , oxidoreduc- tase and cytoskeletal protein accounts majority of identified proteins in molecular function ; the identified proteins mainly participate in protein metabolism and modification, nucleoside, nucleotide and nucleic acid metabolism, cell structure and motility, immunity and defense etc.; regulation of actin cytoskeleton ,focal adhension and antigen processing and presentation etc. perhaps were the key pathways in lymphtic metastasis of tumor.更多还原