【作者】 汪磊；

【导师】 钱旻；

【作者基本信息】 华东师范大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 原发性肝癌是一种分布范围广、危害严重的致死性疾病，我国是肝癌高发地区，每年至少有12万人死于原发性肝癌。然而在临床上对肝癌目前尚缺乏根治性的治疗方法，究其原因主要与我们对肝癌发生、发展的机理还知之甚少，对肝癌细胞具有的特殊生物学特性及其分子机理还有待于进一步深入研究。因此加强肝癌基础理论的研究，提升有效的早期诊断和治疗技术具有重要的理论意义和实际应用价值。本实验的前期工作是利用噬菌体展示肽库技术筛选与肝癌特异性结合的导向肽，已经建立了多种筛选方法，尤其是在荷瘤动物模型中的体内筛选技术，同时建立了一套肿瘤特异性导向肽在体内外靶向作用的鉴定技术。目前已经筛选获得了多条特异性结合于肝癌组织的导向肽，并获得了中国发明专利。本论文在此基础上，拟利用获得的三种肝癌细胞特异性导向肽（A54、WP05和JY96），通过固相生物淘洗和免疫学技术从己构建的肝癌cDNA表达文库中筛选出这些导向肽的结合蛋白基因，并分析这些基因与肝癌发生、发展之间的联系，同时通过亲和分离技术从肝癌组织蛋白提取液中分离与这些导向肽相互作用的结合蛋白。本论文主要分成以下几个部分：一、肝癌导向肽的体外结合活性鉴定在前期工作中我们筛选获得了一系列能够特异性结合肝癌细胞的噬菌体肽，然而体外化学合成的肽是否还能保持这一导向特性还需要进一步验证。我们采用化学合成的方法合成了多肽WP05、JY96和A54，并对其进行羧基荧光素（FAM）标记，通过体外结合活性来验证这些导向肽与肝癌细胞的特异性结合作用。经验证，化学合成的WP05、JY96和A54肽确实能在体外与肝癌细胞特异性结合，为分离导向肽在肝癌细胞上的结合蛋白奠定了理论基础。二、肝癌cDNA表达文库的构建及肝癌导向肽结合蛋白基因的筛选通过cDNA表达文库筛选策略分离肝癌特异性导向肽WP05、JY96和A54的结合蛋白基因。以人肝癌细胞BEL-7402的裸鼠移植瘤为材料，构建了肝癌cDNA表达文库，原始文库滴度为6.1×10⁷pfu／ml，重组率99.3％，扩增后滴度1.4×10¹¹pfu／ml，达到了用于目的基因分离筛选和克隆表达的建库要求。以生物素（Biotin）标记的导向肽为探针，同时采用固相筛淘法和免疫学方法筛选cDNA文库。实验结果显示：利用导向肽WP05从文库中筛选到了SNX27基因；利用导向肽JY96筛选到RBX1基因。三、siRNA抑制SNX27基因表达对肝癌细胞的影响SNX27是通过导向肽WP05从肝癌cDNA表达文库中筛选到的基因。SNX27基因在肝癌细胞内上调表达，因此我们通过RNA干扰技术分析SNX27基因与肝癌发生、发展之间的相互关系。RNA干扰采用siRNA真核表达载体的方法诱导。通过在线siRNA设计工具设计SNX27基因干扰靶序列，利用pAS质粒构建siRNA真核表达载体pAS／SNX27i。通过转染试剂将pAS／SNX27i转染到BEL-7402细胞中，分析RNA干扰抑制效率，分别检验了SNX27基因表达被抑制的肿瘤细胞在增殖、粘附和迁移的变化。结果表明，转染后SNX27基因的表达抑制效率达到了29.5％。SNX27表达抑制导致肿瘤细胞发生如下变化：增殖速率显著下降；细胞粘附性下降，与对照细胞相比差异极显著（P=0.00084＜0.005）；细胞迁移性下降，与对照细胞相比差异显著（0.05＞P=0.0016＞0.001）。四、siRNA抑制RBX1基因表达对肝癌细胞的影响RBX1是通过导向肽JY96从肝癌cDNA表达文库中筛选到的基因。经差异表达分析，RBX1在肝癌细胞BEL-7402中呈上调表达，提示其可能与肝癌有一定联系。构建RBX1基因的siRNA真核表达载体pAS／RBX1i，通过转染试剂将pAS／RBX1i转染到BEL-7402细胞中，检验RBX1基因表达被抑制的肿瘤细胞增殖、粘附和迁移的变化。结果表明，转染后RBX1基因的表达抑制效率达到了68.6％。RBX1表达抑制导致肿瘤细胞发生如下变化：增殖几乎完全停滞；细胞粘附性下降，与对照相比差异极显著（P=0.0003＜0.005）；细胞迁移性下降，与对照相比差异极显著（P=0.0007＜0.005）。四、肝癌蛋白的提取及肝癌导向肽结合蛋白的亲和分离为分离得到肝癌导向肽A54的结合蛋白，我们尝试从肝癌组织蛋白中用亲和分离技术获得A54肽的结合蛋白。从荷瘤裸鼠模型肿瘤组织中提取肝癌组织蛋白。以Biotin-A54肽作为亲和分离介质，通过多种亲和分离技术从提取的肝癌组织蛋白中获得能与A54肽相互作用的结合蛋白。经对洗脱蛋白的质谱进行分析，发现洗脱蛋白为分子量为67kDa的一组蛋白，其中包括已有报导的潜在肿瘤膜标志蛋白HSC71。经生物信息学分析，HSC71蛋白具有能与A54肽相互作用的空间结构。从上面的实验我们可以得到以下结论：1．通过体外的细胞结合实验，证明了导向肽WP05、JY96和A54能够与肝癌细胞特异性结合；2．成功构建了人肝癌细胞裸鼠移植瘤的cDNA表达文库，达到了用于目的基因分离筛选和克隆表达的建库要求；3．利用导向肽WP05从cDNA表达文库中筛选到SNX27基因；4．利用导向肽JY96从cDNA表达文库中筛选到RBX1基因；5．成功构建了SNX27基因siRNA真核表达载体pAS／SNX27i，转染BEL-7402细胞后，对SNX27基因表达抑制效率达到29.5％，能够抑制肿瘤细胞增殖、粘附及其迁移。6．成功构建了RBX1基因siRNA真核表达载体pAS／RBX1i，转染BEL-7402细胞后，对RBX1基因表达抑制效率达到68.6％，能够抑制肿瘤细胞增殖、粘附及其迁移。7．尝试通过亲和分离方法分离导向肽A54的结合蛋白，分离得到的一组67kDa的蛋白，其中包含HSC71。HSC71具有能与A54相互作用的空间结构。更多还原

【Abstract】 Human hepatocellular carcinoma （HCC） is one of the most common and deadly cancers in the world. China is one of the most widespread countries of HCC. At least 120,000 people in China were dead from it every year. However, there are no efficient clinical methods for radical cure of HCC, the reason of which is that we still know little about the mechanisms of HCC carcinogenesis and development and that we should do further studies on the special biological characteristics and molecular mechanisms of HCC. Therefore, it is of high theoretical and practical value to strengthen researches in HCC basic theory and to improve efficient technologies in early diagnosis and therapy.The preceding work of this research is the panning of hepatocarcinoma specific homing peptide by phage display technology. During the preceding research, several panning methods had been established, especially the in vivo panning technology based on nude mice animal model, and a series of identification technologies for detecting the in vitro and in vivo target effect of hepatocarcinoma homing peptide. So far, several specific hepatocarcinoma homing peptides have been gained, and several Discovery Patents of China for them had been obtained. Based on the above work, in this paper, several specific hepatocarcinoma homing peptides we had got （A54, WP05and JY96） were used to screen peptide-binding proteins which is on the HCC cells and can interact with those homing peptides, by several methods such as cDNA library screening and protein-affinity isolation. Then the correlation between those proteins and hepatocarcinogenesis as well as proteins and HCC development was analyzed.This paper can be divided into three parts: A. Identification of hepatocarcinoma homing peptide binding activity in vitro.A series of phage peptides specifically recognizing HCC cells had been obtained during the preceding research. However, it should be further verificated whether the chemically synthesized peptides keeps the homing characteristics. The peptides WP05, JY96, A54 were chemically synthesized and marked with FAM. The specific binding of homing peptides to HCC cells was verificated via the in vivo binding activity of them. Through verification, we found that chemically synthesized peptides WP05, JY96, A54 indeed bind specifically to HCC cells in vivo, which through the theoretical foundation for isolating HCC cell proteins bound by homing peptides.B. Construction of a hepatocarcinoma cDNA expression library and panning of proteins bound by specific HCC homing peptides.Proteins bound by hepatocarcinoma homing peptides WP05, JY96 and A54 were isolated by the strategy of screen of cDNA expression library. A hepatocarcinoma cDNA expression library was constructed using human transplantation tumor bear by nude mice. The titer of original library is 6.1 ×10⁷pfu/ml, recombination rate is 99.3%. While after amplification, the titer grows to 1.4×10¹¹ pfu/ml, which reaches the required criteria for constructing a library to screen and isolate target genes. The cDNA library was screened by homing peptides as probes. Every peptide was used for two kinds of screening, which were solid-phase bio-screening and immunology screening. Gene SNX27 was obtained by screening with WP05 and gene RBX1 was obtained by screening with JY96.C. Effects of SNX27 gene expression inhibition by siRNA on hepatocellular carcinoma.SNX27 is a gene obtained through screening based on homing peptide WP05 and is up-regulation in hepatocarcinoma. The correlation between SNX27 expression and hepatocarcinogenesis as well as HCC development was expected to be analyzed by RNAi. The target sequence of SNX27 siRNA was designed by online software and the eukaryotic expression vector of SNX27 siRNA named pAS/SNX27i was constructed. pAS/SNX27i was transfected into BEL-7402 cells with transfection reagent Sofast^TM and the inhibition effects of RNAi was analyzed. The proliferation, adhesion and migration of cancer cells after SNX27 expression being inhibited were detected. Results show that after transfection, the inhibition rate of SNX27 gene expression reached to 29.5%, cell proliferation was significantly inhibited; cell adhesion was significantly inhibited, different from that of control with P=0.00084<0.005; cell migration was significantly inhibited, different from control with 0.05>P=0.0016>0.001.D. Effects of RBX1 gene expression inhibition by siRNA on hepatocellular carcinoma.RBX1 is a gene obtained through screening based on homing peptide JY96. The correlation between RBX1 expression and hepatocarcinogenesis as well as HCC development was expected to be analyzed by RNAi. The sequence of RBX1 siRNA was designed by online software and the eukaryotic expression vector of RBX1 siRNA named pAS/RBX1i was constructed. pAS/RBX1i was transfected into BEL-7402 cells with transfection reagent Sofast^TM and the inhibition effects of RNAi was analyzed. The proliferation, adhesion and migration of cancer cells after RBX1 expression being inhibited were detected. Results show that after transfection, the inhibition rate of RBX1 gene expression reached 68.6%, the speed of cell proliferation almost totally inhibited; cell adhesion was significantly inhibited, different from that of control with P= 0.0003<0.005 ; cell migration was significantly inhibited too, different from control with P= 0.0007<0.005. E. The study on isolation of proteins bound by specific HCC-binding peptides.To obtain the protein bound by specific hepatocarcinoma homing peptides A54, the proteins from HCC tissue were screened by affinity chromatography. The tumor tissue from nude mice model was used for the extraction of HCC tissue proteins. With biotin-A54 as affinity mediator, proteins, which might interact with A54, were isolated from the protein extraction of HCC tumor tissue. The 67kDa eluate was found to be a mixture of several proteins including a potential tumor membrane marker HSC71. By bioinformatics analysis, we found protein HSC71 has a three-dimensional structure which supporting its interaction with A54. From all the above, we can conclude that:Ⅰ. Hepatocarcinoma homing peptide WP05, JY96 and A54 were identified indeed bind specifically to HCC cells in vivo.Ⅱ. A full-length cDNA expression library for human hepatocarcinoma has been successfully constructed by SMART technology. It reaches the required criteria for constructing a library to screen and isolate target genes.Ⅲ. A gene SNX27 was obtained by screening full-length cDNA expression library with Biotin-WP05 peptide.Ⅳ. A gene RBX1 was obtained by screening full-length cDNA expression library with Biotin-JY96 peptide.Ⅴ. siRNA eukaryotic expression vectors pAS/SNX27i had been successfully constructed. The inhibition rate of gene expression reached 29.5%. The proliferation, adhesion and migration of hepatocarcinoma cell were inhibited after transfection.Ⅵ. siRNA eukaryotic expression vectors pAS/RBX1i had been successfully constructed. The inhibition rate of gene expression reached 68.6%. The proliferation, adhesion and migration of hepatocarcinoma cell were inhibited after transfection.Ⅶ. Affinity chromatography was tried to isolate proteins bound by hepatocarcinoma homing peptide A54. A mixture of 67kDa protein was obtained, including HSC71.HSC71 has a dimensional structure via which HSC71 may interact with A54.更多还原