骨肉瘤荷瘤裸鼠模型的制備及其腫瘤血管內(nèi)皮特異性結(jié)合肽的體內(nèi)篩選
發(fā)布時間：2018-09-19 06:55
【摘要】： 骨肉瘤是最為常見的惡性骨腫瘤,其發(fā)病率占原發(fā)惡性骨腫瘤的22.36%。80～90%的病人在確診時已發(fā)生體內(nèi)其它部位的轉(zhuǎn)移。目前超大劑量化療仍是骨肉瘤最主要的治療方法之一,但化療的同時也會對機體正常組織產(chǎn)生嚴重的毒性作用。因此,增強化療藥物針對骨肉瘤組織的靶向性,成為目前急待解決的一個課題。腫瘤的血管生成是腫瘤發(fā)展的重要環(huán)節(jié),腫瘤必須通過形成新的血管系統(tǒng)來提供足夠的營養(yǎng),以支持其繼續(xù)生長,因此將腫瘤新生血管內(nèi)皮上的某些特異性分子作為藥物作用的新靶點,日益受到研究者的密切關(guān)注。為了獲取能與上述靶點特異性結(jié)合的配體,必須具備有效的篩選手段。噬菌體展示技術(shù)的應(yīng)用為實現(xiàn)此目的提供了一個全新的工具。 噬菌體展示技術(shù)是20世紀90年代發(fā)展起來并得到廣泛應(yīng)用的新技術(shù),其原理是將外源多肽或蛋白與噬菌體的一種衣殼蛋白融合表達,融合蛋白將展示在噬菌體的表面,而編碼這個融合子的DNA則位于該噬菌體內(nèi)。噬菌體展示技術(shù)的一個最基本的特征是將表現(xiàn)型和基因型有效聯(lián)系起來,即噬菌體表面的特定表現(xiàn)型與噬菌體顆粒中的基因型信息相對應(yīng),如需得到某個特定的表現(xiàn)型,只需在噬菌體基因組中插入該表現(xiàn)型的相關(guān)基因即可。噬菌體展示技術(shù)使大量隨機多肽與其DNA編碼序列之間建立了直接聯(lián)系,使各種靶分子(抗體、酶、細胞表面受體等)的多肽配體通過一種被稱為淘選(panning)的體外選擇程序得以快速鑒定。最簡單的淘選程序是將噬菌體展示肽庫與包被有靶分子的平板(或磁珠)共溫育,先洗去未結(jié)合的噬菌體,然后洗脫特異性結(jié)合的噬菌體。將被洗脫的噬菌體進行擴增,然后再進行下一輪的結(jié)合/擴增循環(huán),以富集那些可結(jié)合序列。經(jīng)3～4輪淘選后,通過DNA測序?qū)γ總�可結(jié)合克隆進行定性。展示在噬菌體表面的隨機肽庫可應(yīng)用于許多方面的研究,包括繪制抗原表位圖譜、研究蛋白質(zhì)-蛋白質(zhì)相互作用和鑒定非肽配體的肽模擬物等。 Ph.D.-C7C噬菌體展示肽庫是將隨機七肽融合到M13噬菌體次要衣殼蛋白(pⅢ)上而構(gòu)建成的一個組合文庫。所展示的隨機多肽兩側(cè)各有一個半胱氨酸(Cys)。在非還原條件下,這兩個半胱氨酸自發(fā)地形成一個二硫鍵,使展示的多肽環(huán)化。受限于二硫鍵環(huán)內(nèi)的7肽庫已被證實能識別抗原表位結(jié)構(gòu)、D-氨基酸靶分子的鏡像配基及開發(fā)以多肽為基礎(chǔ)的治療藥物等。 體內(nèi)噬菌體展示技術(shù)更是創(chuàng)造性地將常規(guī)的噬菌體展示技術(shù)與動物模型相結(jié)合,是尋找組織、器官特異性結(jié)合多肽的有效手段。此方法可在受體分子尚不清楚的情況下,以受體天然存在的環(huán)境——組織器官為配基,利用噬菌體短肽的抗原特異性,尋找未知的靶分子,確定其結(jié)構(gòu)域。本研究采用體內(nèi)噬菌體展示技術(shù),在骨肉瘤荷瘤裸鼠模型體內(nèi)進行了腫瘤血管內(nèi)皮細胞的篩選,尋找其表面分子的特異性結(jié)合肽,以期為骨肉瘤的治療及其發(fā)生發(fā)展機制的研究提供新的思路與線索。 我們在BALB/c裸鼠體內(nèi)原位接種鼠源性骨肉瘤細胞UMR-106,成功制作骨肉瘤荷瘤動物模型。在骨肉瘤荷瘤裸鼠模型體內(nèi)進行了腫瘤血管內(nèi)皮細胞的四輪篩選。將第4輪篩選產(chǎn)物鋪板后,隨機挑取60個噬菌體克隆,經(jīng)過PCR擴增出其目的片段以后,進行DNA測序。 我們對出現(xiàn)次數(shù)最多的噬菌體展示七肽(TKPDKGY)進行了體內(nèi)導(dǎo)向效果鑒定和免疫組化染色分析。體內(nèi)導(dǎo)向效果鑒定實驗顯示,呈現(xiàn)TKPDKGY的噬菌體在單位重量腫瘤中的回收量是3.67×10~8 pfu/g,分別是腦組織回收量的32.19倍(1.14×10~7 pfu/g),肺組織回收量的24.14倍(1.52×10~7 pfu/g),腎組織的9.97倍(3.68×10~7 pfu/g)。初步認為該噬菌體克隆在動物模型腫瘤組織內(nèi)有很好的導(dǎo)向效果。 免疫組化鑒定顯示表達TKPDKGY的噬菌體克隆在骨肉瘤組織內(nèi)能夠較好的富集且主要聚集在腫瘤血管內(nèi)壁,在肺組織、腎組織僅有少量的富集而在腦組織內(nèi)沒有發(fā)現(xiàn)噬菌體富集現(xiàn)象。 總之,本研究在靶分子未知的情況下,采用體內(nèi)噬菌體展示技術(shù),在骨肉瘤荷瘤裸鼠模型體內(nèi)進行了腫瘤血管內(nèi)皮的四輪篩選,獲得了與動物模型骨肉瘤血管內(nèi)皮特異性結(jié)合的短肽序列,通過體內(nèi)回輸驗證及免疫組化鑒定,初步認定表達TKPDKGY的噬菌體克隆對動物模型骨肉瘤血管內(nèi)皮有很好的導(dǎo)向性效果。通過以上研究,我們得出以下幾點結(jié)論: 1.成功復(fù)制了骨肉瘤荷瘤裸鼠模型; 2.運用體內(nèi)噬菌體展示技術(shù),篩選到了動物模型骨肉瘤血管特異性結(jié)合肽庫。并且經(jīng)過四輪篩選,噬菌體文庫在動物模型骨肉瘤組織內(nèi)產(chǎn)生了有效富集。 3.隨機挑取60個噬菌體克隆,經(jīng)過PCR擴增目的片段以后,進行DNA測序,得到了3個重復(fù)性較高的短肽序列。 4.對側(cè)序結(jié)果中重復(fù)率最高的3個噬菌體克隆進行體內(nèi)回輸實驗,發(fā)現(xiàn)表達TKPDKGY的噬菌體克隆在單位重量骨肉瘤組織中的回收量遠遠高于其他單位正常組織中的回收量。 5.免疫組化實驗進一步證實表達TKPDKGY七肽的噬菌體特異性結(jié)合于動物模型骨肉瘤腫瘤血管內(nèi)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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R738.1;R-332
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