骨肉瘤荷瘤裸鼠模型的制备及其肿瘤血管内皮特异性结合肽的体内筛选
发布时间：2018-09-19 06:55
【摘要】： 骨肉瘤是最为常见的恶性骨肿瘤,其发病率占原发恶性骨肿瘤的22.36%。80～90%的病人在确诊时已发生体内其它部位的转移。目前超大剂量化疗仍是骨肉瘤最主要的治疗方法之一,但化疗的同时也会对机体正常组织产生严重的毒性作用。因此,增强化疗药物针对骨肉瘤组织的靶向性,成为目前急待解决的一个课题。肿瘤的血管生成是肿瘤发展的重要环节,肿瘤必须通过形成新的血管系统来提供足够的营养,以支持其继续生长,因此将肿瘤新生血管内皮上的某些特异性分子作为药物作用的新靶点,日益受到研究者的密切关注。为了获取能与上述靶点特异性结合的配体,必须具备有效的筛选手段。噬菌体展示技术的应用为实现此目的提供了一个全新的工具。 噬菌体展示技术是20世纪90年代发展起来并得到广泛应用的新技术,其原理是将外源多肽或蛋白与噬菌体的一种衣壳蛋白融合表达,融合蛋白将展示在噬菌体的表面,而编码这个融合子的DNA则位于该噬菌体内。噬菌体展示技术的一个最基本的特征是将表现型和基因型有效联系起来,即噬菌体表面的特定表现型与噬菌体颗粒中的基因型信息相对应,如需得到某个特定的表现型,只需在噬菌体基因组中插入该表现型的相关基因即可。噬菌体展示技术使大量随机多肽与其DNA编码序列之间建立了直接联系,使各种靶分子(抗体、酶、细胞表面受体等)的多肽配体通过一种被称为淘选(panning)的体外选择程序得以快速鉴定。最简单的淘选程序是将噬菌体展示肽库与包被有靶分子的平板(或磁珠)共温育,先洗去未结合的噬菌体,然后洗脱特异性结合的噬菌体。将被洗脱的噬菌体进行扩增,然后再进行下一轮的结合/扩增循环,以富集那些可结合序列。经3～4轮淘选后,通过DNA测序对每个可结合克隆进行定性。展示在噬菌体表面的随机肽库可应用于许多方面的研究,包括绘制抗原表位图谱、研究蛋白质-蛋白质相互作用和鉴定非肽配体的肽模拟物等。 Ph.D.-C7C噬菌体展示肽库是将随机七肽融合到M13噬菌体次要衣壳蛋白(pⅢ)上而构建成的一个组合文库。所展示的随机多肽两侧各有一个半胱氨酸(Cys)。在非还原条件下,这两个半胱氨酸自发地形成一个二硫键,使展示的多肽环化。受限于二硫键环内的7肽库已被证实能识别抗原表位结构、D-氨基酸靶分子的镜像配基及开发以多肽为基础的治疗药物等。 体内噬菌体展示技术更是创造性地将常规的噬菌体展示技术与动物模型相结合,是寻找组织、器官特异性结合多肽的有效手段。此方法可在受体分子尚不清楚的情况下,以受体天然存在的环境——组织器官为配基,利用噬菌体短肽的抗原特异性,寻找未知的靶分子,确定其结构域。本研究采用体内噬菌体展示技术,在骨肉瘤荷瘤裸鼠模型体内进行了肿瘤血管内皮细胞的筛选,寻找其表面分子的特异性结合肽,以期为骨肉瘤的治疗及其发生发展机制的研究提供新的思路与线索。 我们在BALB/c裸鼠体内原位接种鼠源性骨肉瘤细胞UMR-106,成功制作骨肉瘤荷瘤动物模型。在骨肉瘤荷瘤裸鼠模型体内进行了肿瘤血管内皮细胞的四轮筛选。将第4轮筛选产物铺板后,随机挑取60个噬菌体克隆,经过PCR扩增出其目的片段以后,进行DNA测序。 我们对出现次数最多的噬菌体展示七肽(TKPDKGY)进行了体内导向效果鉴定和免疫组化染色分析。体内导向效果鉴定实验显示,呈现TKPDKGY的噬菌体在单位重量肿瘤中的回收量是3.67×10~8 pfu/g,分别是脑组织回收量的32.19倍(1.14×10~7 pfu/g),肺组织回收量的24.14倍(1.52×10~7 pfu/g),肾组织的9.97倍(3.68×10~7 pfu/g)。初步认为该噬菌体克隆在动物模型肿瘤组织内有很好的导向效果。 免疫组化鉴定显示表达TKPDKGY的噬菌体克隆在骨肉瘤组织内能够较好的富集且主要聚集在肿瘤血管内壁,在肺组织、肾组织仅有少量的富集而在脑组织内没有发现噬菌体富集现象。 总之,本研究在靶分子未知的情况下,采用体内噬菌体展示技术,在骨肉瘤荷瘤裸鼠模型体内进行了肿瘤血管内皮的四轮筛选,获得了与动物模型骨肉瘤血管内皮特异性结合的短肽序列,通过体内回输验证及免疫组化鉴定,初步认定表达TKPDKGY的噬菌体克隆对动物模型骨肉瘤血管内皮有很好的导向性效果。通过以上研究,我们得出以下几点结论: 1.成功复制了骨肉瘤荷瘤裸鼠模型; 2.运用体内噬菌体展示技术,筛选到了动物模型骨肉瘤血管特异性结合肽库。并且经过四轮筛选,噬菌体文库在动物模型骨肉瘤组织内产生了有效富集。 3.随机挑取60个噬菌体克隆,经过PCR扩增目的片段以后,进行DNA测序,得到了3个重复性较高的短肽序列。 4.对侧序结果中重复率最高的3个噬菌体克隆进行体内回输实验,发现表达TKPDKGY的噬菌体克隆在单位重量骨肉瘤组织中的回收量远远高于其他单位正常组织中的回收量。 5.免疫组化实验进一步证实表达TKPDKGY七肽的噬菌体特异性结合于动物模型骨肉瘤肿瘤血管内皮。
[Abstract]:Osteosarcoma is the most common malignant bone tumor. The incidence of osteosarcoma is 22.36% of the primary malignant bone tumor. 80-90% of the patients have metastasized to other parts of the body at the time of diagnosis. Therefore, enhancing the targeting of chemotherapeutic drugs to osteosarcoma tissue has become an urgent problem to be solved. Angiogenesis is an important link in tumor development. Tumors must provide adequate nutrition through the formation of new vascular systems to support their continued growth. Therefore, some specificity of tumor neovascularization endothelium is needed. Molecules, as new targets of drug action, have attracted more and more attention from researchers. In order to obtain ligands that can specifically bind to these targets, effective screening methods are necessary.
Phage display technology is a new technology developed and widely used in the 1990s. The principle of phage display technology is to fuse and express exogenous peptides or proteins with a capsid protein of a phage. The fusion protein will be displayed on the surface of the phage, and the DNA encoding the fusion protein is located in the phage. Phage display technology allows a large number of random peptides to be inserted into the phage genome to obtain a specific phenotype. The direct connection between the DNA coding sequences enables the rapid identification of polypeptide ligands of various target molecules (antibodies, enzymes, cell surface receptors, etc.) through an in vitro selection procedure known as panning. The simplest panning procedure is to co-incubate the phage display peptide library with a plate (or magnetic bead) coated with the target molecule and wash it first. Unbound phages are removed, and then specifically bound phages are eluted. The eluted phages are amplified, and then the next round of binding/amplification cycles are performed to enrich those binding sequences. After three to four rounds of selection, each binding clone is characterized by DNA sequencing. The random peptide library displayed on the phage surface is responsive. It has been used in many fields, including mapping antigen epitopes, studying protein-protein interactions and identifying peptide mimics of non-peptide ligands.
Ph.D. -C7C phage display peptide library is a combinatorial library constructed by fusing random heptapeptides into M13 phage secondary capsid protein (pIII). The displayed random peptides have a cysteine (Cys) on each side. Under non-reductive conditions, the two cysteines spontaneously form a disulfide bond, which cyclizes the displayed peptides. Seven peptide libraries within disulfide bonds have been shown to recognize epitope structures, mirror ligands for D-amino acid target molecules, and to develop peptide-based therapeutic drugs.
In vivo phage display technology is a creative way to combine conventional phage display technology with animal models, and is an effective means to search for specific binding peptides to tissues and organs. In this study, in vivo phage display technique was used to screen tumor vascular endothelial cells in nude mice bearing osteosarcoma, and to search for specific binding peptides of their surface molecules. Road and clue.
In BALB/c nude mice, murine osteosarcoma cell UMR-106 was inoculated in situ to establish a tumor-bearing animal model of osteosarcoma. Four rounds of screening of tumor vascular endothelial cells were carried out in the model of osteosarcoma-bearing nude mice. After that, DNA sequencing was performed.
In vivo targeting effect of phage display heptapeptide (TKPDKGY) with the highest number of occurrences was identified and analyzed by immunohistochemical staining. The tissue recovery was 24.14 times (1.52 *10~7 pfu/g) and 9.97 times (3.68 *10~7 pfu/g) of the kidney tissue.
Immunohistochemical staining showed that phage clones expressing TKPDKGY were well enriched in osteosarcoma tissues and mainly concentrated in the inner wall of tumor blood vessels. In lung tissues, there was only a small amount of phage enrichment in kidney tissues, but no phage enrichment was found in brain tissues.
In conclusion, in vivo phage display technique was used to screen the tumor vascular endothelium in nude mice bearing osteosarcoma with unknown target molecule, and the short peptide sequence specifically binding to the vascular endothelium of the animal model osteosarcoma was obtained. Bacteriophage cloning up to TKPDKGY has a good directional effect on the vascular endothelial cells of osteosarcoma animal models.
1. the nude mice model of osteosarcoma was successfully replicated.
2. In vivo phage display technique was used to screen the angiosarcoma-specific binding peptide library of animal models. After four rounds of screening, the phage library was effectively enriched in the osteosarcoma tissues of animal models.
3. Sixty phage clones were randomly selected and amplified by PCR. Three highly repeatable peptides were obtained by DNA sequencing.
4. In vivo transfusion of three phage clones with the highest repetition rate in lateral sequence results showed that the recovery of phage clones expressing TKPDKGY in osteosarcoma tissue per unit weight was much higher than that in other normal tissues.
5. Immunohistochemical assay further confirmed that the phage expressing TKPDKGY heptapeptide specifically binds to the vascular endothelium of osteosarcoma animal model.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R738.1;R-332
[Abstract]:Osteosarcoma is the most common malignant bone tumor. The incidence of osteosarcoma is 22.36% of the primary malignant bone tumor. 80-90% of the patients have metastasized to other parts of the body at the time of diagnosis. Therefore, enhancing the targeting of chemotherapeutic drugs to osteosarcoma tissue has become an urgent problem to be solved. Angiogenesis is an important link in tumor development. Tumors must provide adequate nutrition through the formation of new vascular systems to support their continued growth. Therefore, some specificity of tumor neovascularization endothelium is needed. Molecules, as new targets of drug action, have attracted more and more attention from researchers. In order to obtain ligands that can specifically bind to these targets, effective screening methods are necessary.
Phage display technology is a new technology developed and widely used in the 1990s. The principle of phage display technology is to fuse and express exogenous peptides or proteins with a capsid protein of a phage. The fusion protein will be displayed on the surface of the phage, and the DNA encoding the fusion protein is located in the phage. Phage display technology allows a large number of random peptides to be inserted into the phage genome to obtain a specific phenotype. The direct connection between the DNA coding sequences enables the rapid identification of polypeptide ligands of various target molecules (antibodies, enzymes, cell surface receptors, etc.) through an in vitro selection procedure known as panning. The simplest panning procedure is to co-incubate the phage display peptide library with a plate (or magnetic bead) coated with the target molecule and wash it first. Unbound phages are removed, and then specifically bound phages are eluted. The eluted phages are amplified, and then the next round of binding/amplification cycles are performed to enrich those binding sequences. After three to four rounds of selection, each binding clone is characterized by DNA sequencing. The random peptide library displayed on the phage surface is responsive. It has been used in many fields, including mapping antigen epitopes, studying protein-protein interactions and identifying peptide mimics of non-peptide ligands.
Ph.D. -C7C phage display peptide library is a combinatorial library constructed by fusing random heptapeptides into M13 phage secondary capsid protein (pIII). The displayed random peptides have a cysteine (Cys) on each side. Under non-reductive conditions, the two cysteines spontaneously form a disulfide bond, which cyclizes the displayed peptides. Seven peptide libraries within disulfide bonds have been shown to recognize epitope structures, mirror ligands for D-amino acid target molecules, and to develop peptide-based therapeutic drugs.
In vivo phage display technology is a creative way to combine conventional phage display technology with animal models, and is an effective means to search for specific binding peptides to tissues and organs. In this study, in vivo phage display technique was used to screen tumor vascular endothelial cells in nude mice bearing osteosarcoma, and to search for specific binding peptides of their surface molecules. Road and clue.
In BALB/c nude mice, murine osteosarcoma cell UMR-106 was inoculated in situ to establish a tumor-bearing animal model of osteosarcoma. Four rounds of screening of tumor vascular endothelial cells were carried out in the model of osteosarcoma-bearing nude mice. After that, DNA sequencing was performed.
In vivo targeting effect of phage display heptapeptide (TKPDKGY) with the highest number of occurrences was identified and analyzed by immunohistochemical staining. The tissue recovery was 24.14 times (1.52 *10~7 pfu/g) and 9.97 times (3.68 *10~7 pfu/g) of the kidney tissue.
Immunohistochemical staining showed that phage clones expressing TKPDKGY were well enriched in osteosarcoma tissues and mainly concentrated in the inner wall of tumor blood vessels. In lung tissues, there was only a small amount of phage enrichment in kidney tissues, but no phage enrichment was found in brain tissues.
In conclusion, in vivo phage display technique was used to screen the tumor vascular endothelium in nude mice bearing osteosarcoma with unknown target molecule, and the short peptide sequence specifically binding to the vascular endothelium of the animal model osteosarcoma was obtained. Bacteriophage cloning up to TKPDKGY has a good directional effect on the vascular endothelial cells of osteosarcoma animal models.
1. the nude mice model of osteosarcoma was successfully replicated.
2. In vivo phage display technique was used to screen the angiosarcoma-specific binding peptide library of animal models. After four rounds of screening, the phage library was effectively enriched in the osteosarcoma tissues of animal models.
3. Sixty phage clones were randomly selected and amplified by PCR. Three highly repeatable peptides were obtained by DNA sequencing.
4. In vivo transfusion of three phage clones with the highest repetition rate in lateral sequence results showed that the recovery of phage clones expressing TKPDKGY in osteosarcoma tissue per unit weight was much higher than that in other normal tissues.
5. Immunohistochemical assay further confirmed that the phage expressing TKPDKGY heptapeptide specifically binds to the vascular endothelium of osteosarcoma animal model.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R738.1;R-332
【参考文献】
相关期刊论文 前8条
1 安平,寿成超,孟麟,董志伟;短肽库中血管内皮细胞生长因子受体Flt-1拮抗剂筛选条件的改进[J];北京大学学报(医学版);2001年05期
2 侍立峰;梁志清;;利用噬菌体肽库技术筛选卵巢特异的VEGFR3亲和肽[J];第三军医大学学报;2006年04期
3 吕海,金大地,史占军,景宗森,郑燕芳,陈祥捚;在骨肉瘤细胞上筛选噬菌体干扰素突变体及其临床意义[J];解放军医学杂志;2000年04期
4 武婕,陈惠鹏;噬菌体肽库研究进展[J];军事医学科学院院刊;2000年01期
5 李晶;汪磊;韩红辉;杜冰;钱e，
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