【作者】 张慧珍；

【导师】 吴逸明；

【作者基本信息】 郑州大学 ， 病理与病理生理学， 2005， 博士

【摘要】 肺癌是严重危害人类健康的恶性肿瘤之一,也是目前全世界发病率和死亡率最高的癌症。肺癌的发生、增殖、侵袭是多因素参与、多步骤完成的,虽然从基因水平和转录水平已对肺癌进行了许多成功的研究,但其癌变机制仍不十分明确,也缺乏有效的用于肺癌预警和预后监测的特异分子标志物。因而能够高效率、高通量地筛查组织中与肺癌发生、发展相关蛋白并制备其抗体,在肺癌肿瘤的预警、筛检、预后、治疗中显得极为重要。 本课题应用蛋白质组学技术对肺癌组织与配对的癌旁组织的蛋白质组进行筛选和识别,根据蛋白质表达差异筛选肺癌相关蛋白;运用蛋白质纯化技术从肺癌组织中获得HSP70纯品,并从噬菌体表面展示技术构建的人源性肺癌单链抗体库中初步筛选到融合抗体。对我国高发肿瘤的防治实践有重要的应用价值,对阐明肺癌发生的分子机制和预后研究以及靶向治疗有重要理论意义。 研究方法: 1 蛋白质组学方法筛选鉴定肺癌相关蛋白 1.1 收集16例手术切除的新鲜肺癌组织及同一患者手术的癌旁组织,标本均于液氮中保存备用。用双向电泳蛋白裂解液提取可溶性总蛋白,并用Bradford方法测定蛋白浓度。用于分析型和制备型凝胶上样量的依据。 1.2 应用等电聚焦(IEF)电泳和十二烷基磺酸钠聚丙烯酰凝胶电泳(SDS-PAGE)技术将16例配对的肺癌组织和癌旁组织可溶性总蛋白分离,得到肺癌的蛋白质更多还原

【Abstract】 Lung cancer Is a kind of disease with high incidence and mortality. It’s a complex process involving multiple events and steps, there are many factors participating the development of lung cancer. Though some molecular pathogenesis studies on human lung cancer have been undertaken successfully in gene (DNA) and transcription (mRNA) levels, the carcinogenic mechanism is still unclear. There is no special molecular marker for early-stage diagnosis and prognosis evaluation. So lung cancer associated proteins and their antibodies are very useful in screening and prognosis of lung cancer.Proteomics was used to screen proteins of human cancerous lung tissue and paired normal lung tissue in this study. Lung cancer associated proteins are screened according to the different protein expression maps, and HSP70 is acquired by purifying, we also constructed human lung cancer antibody library using the phage display technique, which is very valuable in prevention and treatment of tumors, and has important theoretical significance to elucidate the molecular mechanism of lung cancer. Methods: 1. Screening and identification of lung cancer associated proteins by proteomicstechnique 1.1 16 fresh lung cancer tissues and paired normal tumor-adjacent tissues of thesame patients are collected. All the tissues are deposited in liquid nitrogen. The soluble proteins are extracted with the lysis solution in two-dimensional gel elcctrophoresis (2DE). and the protein concentration was measured by Bradford’s method.1.2, To obtain 2-DE maps of lung cancer proteins, the soluble proteins were separated by Isoelectric Focusing (IEF) electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) After scanning image using GvS-800 and analyzing data with PDQuest gel image analysis software, we found out the different expression proteins.1,3 The specific proteins were cut out from the gel and then digested by typsin. Peptide mass finger-printer (PMF) was gained by matrixes assisted laser adsorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Then use biology information to search protein database of these PMF and identify the proteins, and analyze the biological significance and function of these proteins, which are the candidate proteins of lung cancer biomarkers.2. Construction and screening of anti-lung-cancer single-chain antibody library2.1 Total RNA were extracted from lymph node of lung cancer patients, and cDNAwere synthesized by RT-PCR. A set of oligonucleotide Primers were designed and synthesized according to Vbase and geneBank. The variable regions of human antibodies were amplified by half-nesting PCR, After digested with restrictive enzyme, they are linked by Linker, and then ligated with pCANTAB 5E. The integrated pCANTAB 5E are transfected into E.coli TG1, and the positive clones are selected by using anmpicillin resistance.2.2 The solution including antibody is prepared, and then concentrated by PEG. Whenthe solution were infected by VKM13 helper phage, ScFv were displayed on the surface of phage.3. Purification of HSP70 and screening antibody library with HSP703.1 We made a column (with 1ml capacity) by using Con A. Before loading the extracted proteins on the column, it was pre-equilibrated. then collected the uncombined proteins.3.2 The proteins were dialyzed overnight in infiltration. After equilibrating DEAEpre-prepared column, dialyzed proteins were loaden on the DEAE ion exchange column ,collected the elution gradiently. Then freeze-dried.3.3 SDS-PAGE was used to make sure every protein’s molecular weight and that near70KD will be HSP70 and mark the peaks, which will be bases for next cycle purification. Then its immunity is identified by Western blot. The antibody library is panning by HSP70 for four times to get enriched antibody library.Results and analysis:1 Screening and Identification of Lung Cancer Associated Proteins by Proteomics Technique1.1 The proteins from 16 pairs of lung cancer tissues were analyzed by 2DE. Theacquired protein maps were very similar, and the expression maps of lung cancer tissue was constructed.1.2 Using PDQuest gel imagination analysis software, we could adjust the picture,detected spots, matched spots, analyzed data.685 spots appeared on the protein map of lung cancer tissues, and 662 spots appeared on the protein map of normal lung cancer-adjacent tissues. There were 523 matching spots on both maps. There were 27 differential protein spots appeared only in the lung cancer tissue. There were 6 protein spots only in the lung cancer-adjacent tissue.1.3 The high abundance lung cancer protein spots were identified and analyzed .The special protein spots were ApolipoproteinA-I precursor (Apo-AI), Peptidyl-prolyl cis-trans isomerase A,Calgranulin B (MRP-14),Calgizzarin (S100C protein),Ferritin light chain (Ferritin L subunit), Ras-related protein Rab-14, apolipoprotein E, Transgelin, UMP-CMP kinase.2. Construction human lung cancer single-chain phage display antibody library Total RNA were extracted from lymph node of lung cancer patients, and cDNA were synthesized by RT-PCR. The variable region genes of human antibodies were amplified by half-nesting PCR, the products amplified in first cycle PCR were 700bp , and then we got VH and VL (about 360 bp) after the second cycle PCR . Integrate single chain variable fragment (ScFv) was formedby linking VL and VH with linker, and then was cloned into display vectors pCANTAB 5E. These vectors were transfected E.Coli TGI, We get primaryQantibody library of phage display with 1.2 X10 capacity. 3 Purification of HSP70 and screening antibody library with HSP703.1 After the extracting liquid of lung cancer geting through ConA, it can be dividedinto two parts. One was ConA combination, the other was no combination.3.2 The no combination part had a light strip on comparatively molecular weight of70KD, and there were several other light strips. The non combination part of proteins were isolated by washing DEAE column gradiently. 6 kinds of proteins are collected respectively, and one of them is defined as Mr 70KD protein through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By using Western-blot it was proved that the 70KD protein is HSP70.The enriched antibody library is obtained through four rounds of panning by HSP70. Conclusions:1. Two-dimensional gel electrophoresis maps for human normal lung tissues and cancerous lung tissues proteins were constructed. With the help of PDQuest image analysis software, different protein spots in two-dimensional electrophoresis gels of cancerous lung tissues were found compared with normal lung tissues. Results showed that 17 protein spots were detected in cancerous lung tissues, 6 protein spots were detected in normal lung tissues. Construction of the maps of difference proteins will helpful for corresponding proteomics database.2. These spots were cut off from Coomassie Brilliant Blue staining gels, digested in gel with L-l-tosylamide-2-phenylethyl chloromethyl ketone (TPCK)-trypsin,identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). Tumor-specific protein spots were identified, including ApolipoproteinA-I precursor (Apo-AI), Peptidyl-prolyl cis-trans isomerase A, Calgranulin B (MRP-14), Calgizzarin (S100C protein), Ferritin light chain (Ferritin L subunit), Ras-related protein Rab-14, apolipoprotein E, Transgelin, UMP-CMP kinase. Among these 10 proteins, thepotential significance of the differential expressions is discussed. 3. Different proteins between cancerous lung tissues and normal lung tissues told us that proteins changed in the progression of cell became cancerous. Some of these proteins involved in cell proliferation cell differentiation, others involved in development of cancer. The analysis of proteins over expressed in lung cancer, and making the proteins serve as tumor markers, will widely used for screening, staging, prognosis, monitoring response to treatment,4 .Total RNA were extracted and the variable region genes of human antibodies wereamplified by nesting PCR, the recombination ScFv lung cancer phage display library with 1.2 X108 capacity was constructed successfully. The titre of the phage was 4 X 1013 .After four rounds of panning, the library was enriched,for the further study. It will be useful for isolating specific ScFv against human lung cancer cell.5 . The HSP70 of lung cancer tissue was separated and purified by fast protein liquidchromatography system (FPLC) rapidly and conveniently. It can be the immunogenecity and a tumor vaccine in the future. As an antigen, HSP70 enriched the phage display library after four rounds of panning.更多还原