【作者】 谈燚；

【导师】 吴浩荣；

【作者基本信息】 苏州大学 ， 外科学， 2010， 博士

【摘要】 第一部分胆囊癌患者血清及正常人血清的蛋白质组学分析目的应用比较蛋白质组学方法研究胆囊癌患者及正常人血清之间差异表达的蛋白质,筛选胆囊癌血清标志物以及可能与胆囊癌发病相关的蛋白质。方法利用双向凝胶电泳(2-DE)对6例胆囊癌患者及6例正常人血清的总蛋白进行分离,并用质谱仪对两组间差异蛋白质点进行肽指纹谱和串联质谱鉴定。利用蛋白质印迹分析和免疫组化法检测差异蛋白在胆囊癌患者和健康人血清及组织的表达。结果共有24个蛋白点被成功鉴定。其中在胆囊癌患者血清中高表达的有12个,它们是Splicing factor 3B subunit 5,Cystatin-B,S100A10 Protein,Histone H2B type 2-E, Profilin-1,Eukaryotic translation initiation factor 1A,Isoform 1 of Eukaryotic translation initiation factor 5A-1,FERM domain containing 3 , haptoglobin protein , Glyceraldehyde-3-phosphate dehydrogenase,Serum amyloid P-component precursor,harmonin isoform b3。在胆囊癌患者血清中表达降低的有12个,分别是Apolipoprotein A-I precursor,Myosin regulatory light chain 2,Isoform 1 of Protein canopy homolog 2 precursor,Proteasome subunit beta type-2,ARHGDIA,Superoxide dismutase [Mn],Isoform 1 of Ficolin-3 precursor,zinc alpha-2-glycoprotein 1,HP haptoglobin isoform 2 preproprotein,CD5 antigen-like precursor,CLU clusterin isoform,ITIH4 71kDa protein。Western印迹及免疫组化证实了S100A10蛋白和结合珠蛋白(haptoglobin protein)在胆囊癌患者血清及组织中高表达。结论以2-DE结合质谱鉴定是血清差异蛋白质组学研究的可靠平台。本实验所得的S100A10等差异表达蛋白可能是潜在的诊断胆囊癌的分子标志物或控制肿瘤生长的治疗靶点。第二部分人胆囊癌组织及胆囊良性组织的蛋白质组学分析目的应用比较蛋白质组学方法研究人胆囊癌组织和胆囊良性组织之间差异表达蛋白质。方法利用双向凝胶电泳(2-DE)对6例胆囊癌组织和6例胆囊良性组织的总蛋白进行分离,并用质谱仪对两组间差异蛋白质点进行肽指纹谱和串联质谱鉴定。利用蛋白质印迹分析和免疫组化法检测差异蛋白在胆囊癌组织和胆囊良性组织的表达。结果共有17个蛋白点被成功鉴定。其中在胆囊癌组织中高表达的有9个,它们是AnnexinA3, Phosphatidylethanolamine-binding protein 1(PEBP1),ACTG1 protein,GAPDH,Alcohol dehydrogenase,Fructose-bisphosphate aldolase,Interferon-induced protein with tetratricopeptide,acyl-CoA dehydrogenase,TTR protein;在胆囊癌组织中表达降低的有8个,分别是Superoxide dismutase,Plasma retinol-binding protein precursor,Alpha-crystallin B chain,Hemoglobin,Cathepsin D precursor,Aldo-keto reductase family 1 member B10,Tripartite motif-containing 45,Serotransferrin precursor。Western blot和免疫组化结果显示差异表达蛋白AnnexinA3和Phosphatidylethanolamine-binding protein 1(PEBP1)在胆囊癌组织中表达上调,与2-DE结果一致。结论以2-DE为基础的蛋白质组学技术是肿瘤研究的一种重要手段。本实验所得的AnnexinA3、PEBP1等差异表达蛋白可能成为潜在的诊断胆囊癌的分子标志物或控制肿瘤生长的治疗靶点。第三部分AnnexinA3-miRNA对胆囊癌细胞的生物学效应的研究目的构建针对AnnexinA3的miRNA真核表达载体,并导入胆囊癌细胞,探讨其对AnnexinA3基因的干扰作用以及对胆囊癌细胞生物学效应。方法人工合成AnnexinA3-miRNA的寡核苷酸链,将其插入到pcDNA?6.2-GW/EmGFPmiR载体中;采用脂质体转染法将构建重组体导入人胆囊癌细胞系SGC-996中;采用实时荧光定量PCR(Real-Time PCR)、免疫印迹法(Western blot)分别检测转染细胞AnnexinA3mRNA和蛋白的表达变化。采用细胞计数法检测实验组和对照组SGC-996细胞的增殖;用流式细胞术(FCM)检测转染AnnexinA3-miRNA载体的SGC-996细胞周期及凋亡;用细胞划痕实验观察各组SGC-996细胞的随意运动能力的变化;利用Transwell小室测定转染AnnexinA3-miRNA载体的SGC-996细胞的穿膜侵袭力。结果成功构建了AnnexinA3的miRNA真核表达载体AnnexinA3-miRNA;转染AnnexinA3-miRNA载体后,SGC-996细胞AnnexinA3 mRNA和蛋白表达均较对照组明显下降(p<0.05)。实验组SGC-996细胞增殖较对照组明显减慢(p<0.05)。干扰前后细胞周期无明显差异(p>0.05),但细胞凋亡明显增加(p<0.05)。干扰后胆囊癌细胞迁移能力明显下降(p<0.05)。实验组穿过基底膜的侵袭细胞数量明显少于对照组(p<0.01)。结论AnnexinA3-miRNA真核表达载体构建成功,转染SGC-996细胞后获得稳定表达,并可特异性封闭AnnexinA3的表达。该载体不仅可抑制SGC-996细胞增殖,诱导SGC-996细胞凋亡,而且还可显著抑制SGC-996细胞的侵袭力,为胆囊癌基因治疗研究提供了实验依据。更多还原

【Abstract】 Part I Proteome analysis of human gallbladder cancer serum and normal human serumObjective To compare the serum proteome difference of gallbladder carcinoma patients and healthy subjects, and to provide experimental evidence for finding serum biomarkers of gallbladder cancer. Methods Proteomes of 6 pairs of clinical gallbladder cancer serum samples and normal human serum samples were obtained by two-dimensional gel electrophoresis(2-DE). Comprehensive analyses of proteins were focused on total protein spots exhibiting statistical alternations between the two groups. Protein identification was done by peptide mass fingerprinting with tandem mass spectrometry(MS). Western blot and immunohistochemistry(IHC) were used to detect the expression levels of differential protein S100A10 and haptoglobin in an independent series of serum and tissue samples. Results A total of 12 protein spot-features were found to be significantly increased and 12 significantly decreased in gallbladder cancer serum, including: Splicing factor 3B subunit 5, Cystatin-B, S100A10 Protein, Histone H2B type 2-E, Profilin-1, Eukaryotic translation initiation factor 1A, Isoform 1 of Eukaryotic translation initiation factor 5A-1, FERM domain containing 3, haptoglobin protein, Glyceraldehyde-3-phosphate dehydrogenase, Serum amyloid P-component precursor, harmonin isoform b3, Apolipoprotein A-I precursor, Myosin regulatory light chain 2, Isoform 1 of Protein canopy homolog 2 precursor, Proteasome subunit beta type-2, ARHGDIA, Superoxide dismutase [Mn], Isoform 1 of Ficolin-3 precursor, zinc alpha-2-glycoprotein 1, HP haptoglobin isoform 2 preproprotein, CD5 antigen-like precursor, CLU clusterin isoform, ITIH4 71kDa protein. The increased levels of S100A10 and haptoglobin protein found to be associated with gallbladder cancer was further confirmed by Western blot and immunohistochemistry analysis.Conclusion These results suggest that the combination of 2-DE with MS provides an effective strategy to discover differentially expressed proteins in gallbladder cancer which may be molecular markers for diagnosis or therapeutic targets.Part II Proteome analysis of human glabbladder cancer tissue and benign gallbladder tissueObjective To find out potential molecular targets for gallbladder carcinoma diagnosis and treatment by analyzing and comparing the proteins expressed in human gallbladder carcinoma tissue and benign gallbladder tissue.Methods Proteomes of 6 pairs of glabbladder cancer tissue samples and benign gallbladder tissues were analyzed by two-dimensional gel electrophoresis(2-DE). Comprehensive analyses of proteins were focused on total protein spots exhibiting statistical alternations between the two groups. Protein identification was done by peptide mass fingerprinting with mass spectrometry(MS). In addition, Western blotting and immunohistochemistry (IHC) were performed to examine the expression of certain candidate proteins in an independent series of samples. .Results Protein extracts of individual sample in each type of tissues were separated on two-dimensional gels. Seventeen proteins were successfully identified by MS, in which nine proteins were overexpressed in tumors and the other eight proteins were underexpressed. Western blotting and IHC further validated up-regulated expressions of two candidate protein in tumorous tissues: AnnexinA3 and PEBP1.Conclusion These results suggest that the combination of 2-DE with MS provides an effective strategy to discover differentially expressed proteins in gallbladder cancer which may be molecular markers for diagnosis or therapeutic targets. Part III Study of Biological Effects of AnnexinA3-miRNA in Gallbladder Cancer CellsObjectives To construct recombinant interfering RNA(miRNA) plasmid vector targeting annexinA3, and observe its impact on the growth and apoptosis of human gallbladder cancer cell line SGC-996 in vitro. Methods An annexinA3-miRNA targeting human annexinA3 mRNA common sequence was synthesized and it was inserted into pcDNA?6.2-GW/EmGFPmiR vector. The recombinant plasmid was transfected into SGC-996 cell by Lipofectin. The proliferation of SGC-996 cell was assessed by cell counting in experimental and control groups. The cell cycle and apoptosis of SGC-996 cells was observed by flow cytometry(FCM). The migratory and invasive ability of these cells was detected by scratch-wound healing assay and Transwell.Results The recombinant plasmids containing annexinA3 miRNA were successfully constructed. The expressions of annexinA3 mRNA and protein in SGC-996 cells of experimental groups were significantly decreased,compared to controls(p<0.05). The proliferation of SGC-996 cells in experimental groups was inhibited, compared to controls(p<0.05). FCM analysis showed that the cell cycle had no change, but annexinA3-miRNA can promote apoptosis(p<0.05). AnnexinA3 knockdown cells (pcDNA6.2-miR) exhibited significant decrease in migration. The number of migrated SGC-996 cells in experimental group was far less compared with controls(p<0.01). Conclusions It indicated that miRNA eukaryotic expression vector for annexinA3 would be successfully established. The administration of annexinA3-miRNA in SGC-996 cells can down-regulated annexinA3 expression, inhibit proliferation, induced apoptosis, and also inhibit the migratory and invasive ability. It suggested that miRNA-based targeting annexinA3 strategy might build the experimental foundation for the research of gene therapy in gallbladder cancer.更多还原