【作者】 贺莉；

【导师】 吴开春； 韩英；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 硕士

【摘要】 【背景】肿瘤血管抑制治疗是近年兴起的一种具有良好前景的治疗恶性肿瘤的方法。目前已发现许多具有血管抑制功能的分子,但是临床效果并不令人满意,主要问题是这些分子的肿瘤血管靶向性不强。因此,进一步研究肿瘤环境中血管内皮细胞的特性和分子异质性,发现肿瘤血管内皮细胞差异表达的分子,对于探讨肿瘤血管生成的机制,发展肿瘤血管抑制治疗具有重要意义。噬菌体展示肽库技术为获得选择性靶向到特定器官的小肽提供了方法。利用随机噬菌体环七肽库,我们实验室于2004年在活体内获得了能特异性结合到人胃癌血管内皮细胞的一个环肽CGNSNPKSC,命名为GX1(专利号ZL200410026137.0)。初步实验结果表明,该肽段与人胃癌和结肠癌血管能够特异性结合,而在对照组织脾脏、血管瘤、肝癌组织上未见明显结合;能特异性的与人脐静脉内皮细胞(HUVECs)结合,而与SGC-7901, Eca-109, LoVo及Hep-G2等肿瘤细胞无特异结合;其空间结构相对稳定,具备作为抗原结合表位的特点。将重组质粒编码的GX1-rmhTNFα融合蛋白注入裸鼠体内,发现该融合蛋白能够靶向定位到肿瘤组织,抑制肿瘤生长,并有较低的全身毒副作用。利用活体示踪技术及影像学的方法证明GX1具有良好的体内肿瘤组织的靶向性。以上的实验结果表明GX1能够选择性的靶向到肿瘤血管上,有可能成为肿瘤血管靶向性治疗的特异性小肽。【目的】筛选和鉴定肿瘤血管靶向肽GX1的结合受体,为了进一步阐明GX1的功能和机制奠定基础。【方法】(1)化学合成生物素标记的GX1及无关对照肽;(2)培养内皮细胞并收集细胞膜蛋白;(3)利用免疫沉淀的方法进行免疫磁珠分离,富集能与血管特异性短肽GX1结合的蛋白质;(4)利用生物素-亲和素系统进行western blot免疫检测,筛选出能和GX1结合的目的条带;(5)利用液相色谱-二级质谱(LC-MS/MS)对能与血管特异性短肽GX1结合的蛋白质进行测序鉴定;(6)利用点印迹及竞争抑制实验的方法,验证肽段的特异性结合。【结果】(1)合成的生物素标记的GX1与生物素标记的对照肽,经色谱鉴定,纯度达到了90%。(2)在相差显微镜下,原代培养的内皮细胞呈单层生长,细胞为多边形,边界不清。VIII因子和CD31呈阳性染色。(3)利用western blot的方法,对FLT-1和KDR进行免疫检测,证明提取物中富集了内皮细胞的膜蛋白。(4)利用免疫沉淀和western blot的方法,获得了能与GX1结合的蛋白条带,其分子量为13KDa。(5)利用LC-MS/MS及生物信息学分析,获得了20个候选蛋白,其中9个(45%)为膜蛋白,9个(45%)亚细胞定位不清楚。(6)利用点印迹及竞争抑制实验的方法,得到了能特异与GX1结合的三个蛋白: CDNA FLJ40018fis, clone STOMA2006398 ,diacylglycerol kinase iota和homologues to ankyrin repeat domain 20A。【结论】结合对GX1已有资料的分析,初步将CDNA FLJ40018fis, clone STOMA2006398和diacylglycerol kinase iota作为GX1受体的候选蛋白,该结果为寻找血管抑制治疗的靶标奠定了一定的实验基础。更多还原

【Abstract】 【Background】Tumor anti-angiogeneis is a prospect method for the treatment of malignant tumors in recent years. Many anti-vascular moleculars have been found, but the clinical effects of these moleculars were not satisfied. The main problem is that these moleculars have poor capacity of binding the tumor vascular. Therefore, further researches on tumor vascular endothelial cells and their molecular heterogeneity as well as their differentially expressed molecular markers will have great significance for the study of the mechanism of tumor angiogenesis and the development of the anti-angiogenesis therapy.Phage display peptide libraries have enabled the discovery of peptides that selectively target specific organs. Using a Ph.D.-C7CTM Phage display peptide library, we identified a specific cyclic peptide GX1 that could specifically link to vasculature endothelial cells of human gastric cancer in vivo. Preliminary results showed that this phage peptide could bind to the vascular of human gastric and colon cancers specifically, while distinct binding were not found in spleen, hemangioma and hepatocelluar carcinoma; this peptide was observed only specifically bind to HUVECs, but not to SGC-7901, Eca-109, Lo-Vo and Hep-G2 cells. Its space structure is relatively stable and it has a combination of antigen epitope characteristics. GX1-rmhTNF fusion protein, which had lower systematical toxicity, could be targeted to the tumor tissue and inhibited tumor growth when injecting its recombinant plasmid encoding protein into nude mice. Using tracer technique in vivo and imaging method it has been proved that GX1 had a good capacity of targeting to tumor tissue in vivo. Preliminary experiments suggested that GX1 peptide could selectively target to tumor vascular endothelium and may be as a specific small peptide for tumor targeted therapy.【Aims】Screening and identification of the receptors of GX1 (CGNSNPKSC) peptide binding to tumor vascular endothelium.It may provide some experimental basis for clarifing its functions and mechanisms.【Methods】(1) Chemical synthesized of biotin-labeled GX1 peptide;(2) Cultured endothelial cells and collected membrane proteins;(3) Separated and enriched proteins that specifically bound to short peptide GX1 by using immunoprecipitation method;(4) Screened out the zone that could bind to GX1 by using biotin-avidin system and western blot;(5) Identified the protein band by tandem mass spectrometry (LC-MS/MS) and analyzed it with bioinformatics;(6) By using high throughput peptide synthesis and membrane probing, we found spots that specifically bind to the GX1 peptide.【Results】 (1) Synthesized of biotin-labeled GX1 peptide and biotin-labeled control peptide, the purity reached 90%.(2) By phase contrast microscopy, the cultured endothelial cells grew in confluent monolayers and cells were polygonal with indistinct cell borders. Factor VIII and CD31 antigen determination of endothelial cells were positive.(3) The membrane extracts of HUVECs are enriched with membrane proteins, as shown by western blotting analysis with polyclonal antibodies of FLT-1 and KDR.(4) By using immunoprecipitation and western blot, one band in the position of protein bands 13 KDa was found to bind selectively to the GX1 peptide.(5) Using LC-MS/MS and bioinformatics analysis, we obtained 20 candidate proteins, including 9 (45%) membrane proteins and 9 (45%) subcellular localization unclear proteins.(6) Using point blot and competitive inhibition experiment, three spots were found specifically bind to the GX1 peptide. They were cDNA FLJ40018fis, clone STOMA2006398, diacylglycerol kinase iota and homologues to ankyrin repeat domain 20A.【Conclusions】Diacylglycerol kinase iota and cDNA FLJ40018fis, clone STOMA2006398 could specifically bind to the GX1 peptide. They may be the GX1’s receptors and may provide some experimental basis for probing into new targets of anti-vascular therapy.更多还原