三叶因子在结直肠癌组织中的表达与临床关系及三叶因子-2在大肠杆菌中的重组表达和活性研究
文献类型：学位论文
| 作者 | 黄尤光 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 结直肠癌 临床病理 三叶因子-1 三叶因子-3 QRT-PCR 免疫组织化学 三叶因子-2 大肠杆菌 细胞迁移活性 |
| 其他题名 | The expression of trefoil factors in colorectal cancer with clinic pathology procession and overexpression of hTFF2 in E. coli and its invitro activity |
| 中文摘要 | 癌症是世界发达国家和许多发展中国家人口的疾病主要死亡原因之一，其中，每年结直肠癌的新增病例和死亡病例排在癌症的第三位。在我国，北京、上海等地的统计资料显示，结直肠癌的发病上升很快 ，已排在癌症的第二位。结直肠癌的发生发展过程涉及一系列细胞和分子事件的改变，包括基因结构的异常和基因表达谱的异常。三叶因子（trefoil factor, TFF）是在上世纪80年代末到90初初由不同研究小组先后发现的含特殊的三叶因子结构域的蛋白多肽，其结构域的特征是含38-40个氨基酸残基的肽段中，有6保守的个半胱氨酸残基以1—5、2—4、3—6的方式形成二硫键，从而形成紧密的三叶结构域。在哺乳动物体内目前发现的三叶因子有三种，由粘膜组织内不同细胞合成并分泌到粘膜表面，对粘膜起保护作用。在粘膜损伤时，可通过多种途径促进上皮细胞迁移和抑制细胞凋亡，并促进血管形成，参与粘膜损伤的修复和重建。三叶因子在肿瘤组织中表达，则可能对癌症的发展起促进作用。研究资料显示，三叶因子在肿瘤中的表达异常与多种肿瘤的发生和发展过程有关。我们通过DNA测序检测结直肠癌组织中TFF1和TFF3基因各外显子的核苷酸序列，以确定是否存在基因突变。并用QRT-PCR和免疫组织化学的方法检测结直肠癌组织中TFF1和TFF3的mRNA和蛋白质的表达水平，分析其表达与结直肠癌的临床和病理特征之间的关系。同时，用ELISA方法检测结直肠癌患者血清中TFF1和TFF3的含量，以分析其与临床的关系，并逐步研究这两种三叶因子有否可能作为结直肠癌有用的血清分子标记。 目前得到以下研究结果：①在TFF1基因5`-端非翻译区位于起始密码上游—2bp处有一高频率的（C→T）突变位点,频率为40%，在其他非编码区也发现若干个较低频率的突变位点。未发现TFF3的基因突变；②TFF1和TFF3的mRNA水平在不同患者结直肠癌组织中的表达水平差异很大。与临床病理关系由于样品例数较少，未作统计学出理。结直肠癌组织中TFF1和TFF3蛋白表达检出阳性率分别为90%和94%。TFF1的表达与结直肠癌临床及病理类型未发现统计学意义，TFF3的表达上调与肿瘤淋巴结转移有关；③结直肠癌患者血清中TFF1的含量为（78.6575±53.300ng/ml），比健康人群血清TFF1含量（19.6457±5.3880ng/ml），增高约4倍，这一结果属首次报道。结直肠癌患者血清中TFF3的含量为（27.96±21.985ng/ml），比正常人群血清TFF3含量（9.0875±2.0315ng/ml）增高约3 1 倍。TFF1和TFF3能否作为结直肠癌的血清分子标志，尚需完善相关资料和作进一步研究。 TFF1和TFF3分别含一个三叶结构域，在靠近C-末端有一个游离的半胱氨酸巯基，TFF1和TFF3通过此二硫键形成同源二聚体，是其活性的主要形式。TFF2含两个三叶结构域，在三叶结构域外其靠近N-端和C-端各有一个半胱氨酸，两者以二硫键相连，形成紧密的结构。我们用pET系统克隆和表达人TFF2(hTFF2)，以及TFF2三叶结构域外二硫键解开的突变型TFF2(MhTFF2)，并测定细胞迁移活性。结果获得高效表达的hTFF2和MhTFF2，占细胞质总蛋白量的40%以上，经亲和层析后得到样品纯度在95%以上。对HCT116细胞株的划痕试验表明，hTFF2和MhTFF2对HCT116细胞具有迁移作用，细胞迁移数约为对照BSA的1.5倍。 结论:①结直肠癌组织中三叶因子-1和三叶因子-3基因突变不是三叶因子表达异常的主要原因；②TFF1和TFF3的转录水平在不同结直肠癌组织中有很多差异，TFF3蛋白的高表达与结直肠癌淋巴转移有关；③血清中TFF1和TFF3的含量检测可能会成为结直肠癌有用的血清分子标志；④pET质粒系统可高效表达可溶性三叶因子-2，并可表达获得有细胞迁移活性的重组融合蛋白TFF2；⑤TFF2的三叶结构域外的二硫键对TFF2的细胞迁移活性不是必须的。 |
| 英文摘要 | Cancer is one of leading causes of disease death in developed countries and many developing countries, and new cases and deaths of colorectal cancer have ranked third in the various types of cancer. Statistics show that the incidence of colorectal cancer rapidly rises and has been ranked second in all types of cancer in Beijing and Shanghai, China. The development and progression of colorectal cancer involve in a series of cell and molecular changes, including the abnormal gene structure and gene expression abnormalities. Trefoil factor (TFF) is small protein peptide found with the special domain of the protein trefoil factor by different research groups in the late 80s to early 90. The domain is characterized by a peptide containing 38 -40 amino acid and including six conserved cysteine residues to form disulfide bonds in 1-5,2-4,3-6 way to create a close three-leafed structure . Present study has found that there are three trefoil factors in mammals, which are synthesized by different cells within the mucosal tissues and are secreted into the mucosal surface to protect the mucosal. When the mucosal injury, TFF involve in the repair and reconstruction of injury mucosal by promoting epithelial cell migration, inhibiting apoptosis and promoting angiogenesis through various channels. Trefoil factors may promote the development of cancer when they express in tumor tissue. Studies have shown that trefoil factors abnormal expression in tumor may be related to the occurrence and development process of many cancers. We detected TFF1 and TFF3 gene exons sequence in colorectal cancer tissues through DNA sequencing to definite whether mutations. With QRT-PCR and immunohistochemistry we also definited TFF1 and TFF3 mRNA and protein expression levels in colorectal cancer tissues and analyzed the relationship between their expression and colorectal cancer clinical and pathological features. Meanwhile, we used ELISA to detected serum levels of TFF1 and TFF3 in colorectal cancer patients, and analyzed their clinical relations, and gradually investigated whether the two trefoil factors may be useful as serum markers of colorectal cancer. And we got the following results: ①We found a high frequency (C →T) mutation locating upstream of-2bp of the start codon in the TFF1 gene 5 - untranslated region. Its Mutation frequency was 40%. In other non-coding regions were also found several mutations with lower frequency. However, TFF3 gene mutation was not found; ②Our results indicated that the mRNA levels of TFF1 and TFF3 in colorectal cancer tissues varied greatly in different patients. And its relation to Clinical Pathology was without a reasonable statistics because of the less sample cases. TFF1 and TFF3 protein expression positive rate were 90% and 94% in colorectal cancer tissues respectively. We also found there was no statistical significance between TFF1 expression and clinical and pathological types of colorectal cancer, and up regulation of TFF3 expression was related to lymph node metastasis; ③Our results also displayed that patients with colorectal cancer demonstrated an approximately four-fold increase in plasma levels of TFF1 compared 3 with healthy people, which was the first report. The serum level of TFF3 in colorectal cancer patients was three times higher than that of the normal population. Whether TFF1 and TFF3 can be used as serum markers of colorectal cancer still need to improve the related information and for further study. TFF1 and TFF3 include one trefoil domain respectively, containing a free cysteine thiol near the C-terminal. TFF1 and TFF3 homodimer formation through the disulfide bonds is the main form of its activity. TFF2 contains two trefoil domains, and in the extraterritorial trefoil structure near the N-terminal and its C-terminal have a cysteine respectively. The two cysteines linked by disulfide bonds to form a compact structure. We used PET system to clone and express human TFF2 (hTFF2), as well as extra-territorial structure of TFF2 trefoil unlock disulfide mutant TFF2 (MhTFF2), and detected cell migration activity. The results were highly expressed in hTFF2 and MhTFF2, cytoplasmic total protein accounted for more than 40%. Purified sample was obtained by affinity chromatography and the sample purity was more than 95%. Scratch test on the HCT116 cell line experiment showed that hTFF2 and MhTFF2 had migrating effect on HCT116 cells, the cell migration number was about 1.5 times as the control group of BSA. |
| 语种 | 中文 |
| 公开日期 | 2010-11-12 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6509]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 黄尤光. 三叶因子在结直肠癌组织中的表达与临床关系及三叶因子-2在大肠杆菌中的重组表达和活性研究[D]. 北京. 中国科学院研究生院. 2010. |
入库方式： OAI收割
来源：昆明动物研究所
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