人骨肉瘤细胞U2OS靶向肽的筛

发布时间：2018-05-29 16:51

  本文选题：骨肉瘤 + 噬菌体文库　； 参考：《广州中医药大学》2017年硕士论文

【摘要】：骨肉瘤是一种好发于青少年的恶性骨肿瘤。现阶段主流的治疗手段是术前大剂量化疗、根治性切除及术后继续大剂量化疗。自上世纪八十年代以来,骨肉瘤患者总生存率长期处于平台期,原发性骨肉瘤患者五年生存率约为60%-70%。现阶段骨肉瘤的治疗受限于缺乏能直接将药物递送到肿瘤细胞的靶向载体。筛选与鉴别肿瘤细胞表面受体的特异性配体,是目前肿瘤研究的热点,新兴的噬菌体展示技术是这一领域的强大工具,将可能促进肿瘤诊断和靶向治疗的发展。目的:利用噬菌体展示技术筛选并鉴定对人骨肉瘤U2OS细胞具有高度亲和力和特异性的多肽,进一步将靶向多肽与金纳米笼偶联,评价多肽靶向热疗骨肉瘤细胞的能力(图1),为进一步开发人骨肉瘤的靶向治疗提供基础。方法:1.以U2OS作为靶细胞,人成骨细胞hFOB1.19为阴性消减细胞,对Ph.D.-12噬菌体展示文库进行全细胞差减筛选。首先将噬菌体文库与hFOB1.19细胞充分结合,排除与hFOB1.19结合,即非特异性结合靶细胞的部分噬菌体,将剩下的文库与U2OS充分结合,洗涤后去除特异性与亲和力较低的噬菌体,通过洗脱及酸化裂解得到对U2OS具有较高特异性和亲和力的噬菌体(包括"细胞表面结合噬菌体"及"内化噬菌体"),将这一轮得到的肽库投入下一轮的筛选,经过四轮重复筛选提高筛选的精确性;2.将每一轮得到的噬菌体涂板进行蓝斑计数,随后从计数平板中随机挑选分离良好的单克隆噬菌体,扩增后送测序公司进行序列测定,获得各噬菌体外源多肽的DNA序列;3.将上述重复率1的单克隆噬菌体扩增、纯化,再分别投入筛选(方法同步骤1,"单克隆噬菌体"代替"噬菌体文库")进行反向鉴定;4.挑选重复率较高的单克隆噬菌体,以hFOB1.19、人骨肉瘤细胞MG-63及人乳腺癌细胞MCF-7为对照细胞进行ELISA检测、免疫组织化学染色鉴定其对靶细胞的亲和力及特异性;5.挑选与U2OS特异性及亲和力最高的单克隆噬菌体为目标噬菌体,依据其DNA序列合成目标多肽和FITC标记的目标多肽;6.梯度单位FITC荧光标记的目标多肽分别与靶细胞、hFOB1.19、MG-63及MCF-7结合,通过流式细胞仪检测荧光值鉴定目标多肽对靶细胞的亲和力及特异性;7.靶细胞U2OS与梯度目标噬菌体孵育后,与定量FITC标记的目标多肽孵育,流式细胞仪检测平均荧光强度;U2OS与梯度目标多肽孵育,然后与目标噬菌体孵育,随后涂板进行蓝斑计数,综合以上结果验证目标噬菌体与其外源多肽竞争性结合U2OS;8.将目标多肽与金纳米笼(Goldnanocages,GNCs)偶联形成多肽-金纳米笼偶联物(Peptide-GNCs,P-GNCs),以hF0B1.19为对照细胞,与U2OS充分培养后,808nm近红外激光照射,记录照射前后细胞培养液温度变化,CCK-8法检测细胞增殖、钙黄绿素染色检测细胞死亡情况。结果:1.经过4轮体外筛选后,细胞表面结合噬菌体富集了约144倍(由1.01× 106 pfu增长至1.45×108pfu),内化噬菌体富集了约15.53倍(由8.95×104pfu增加至1.39×106 pfu);2.分别随机挑选第二、三、四轮筛选后的噬菌体81、123、200个,共404个噬菌体进行测序,并按顺序命名为Phage OS-1、Phage OS-2、…Phage OS-404,相应外源多肽命名为Peptide OS-1、Peptide OS-2,…Peptide OS-404,统计各噬菌体重复率,得到重复率较高的细胞表面结合及内化噬菌体各6个;3.单克隆噬菌体富集率实验结果表明:各噬菌体富集率与测序重复率相符;4.ELISA结果显示:Phage OS-7对U2OS亲和力最高,对MG-63亲和力同样较高,与hFOB1.19、MCF-7不结合,为本次研究的目标噬菌体;5.与人骨肉瘤癌旁正常组织切片对比,Phage OS-7在人骨肉瘤组织切片染色呈显著阳性,而野生型噬菌体染色结果为阴性;6.多肽亲和力实验显示:PeptideOS-7(多肽序列为:APWTEAYWWHLP)对U2OS亲和力最高,且随着浓度的增加而升高,对MG-63亲和力同样较高,对hFOB1.19、MCF-7不结合;7.噬菌体及相应多肽竞争性结合实验表明:Peptide OS-7与Phage OS-7竞争性结合U2OS;8.近红外激光照射实验显示:Peptide OS-7-GNCs的光热治疗效果明显优于单纯金纳米笼组。结论:我们通过噬菌体展示技术筛选及鉴定的多肽序列为:APWTEAYWWHLP,该多肽对人骨肉瘤细胞U2OS具有高度亲和力和特异性。本研究提供了通过噬菌体展示技术筛选及鉴定人骨肉瘤细胞U2OS特异性结合多肽的明确实例,以及此类多肽在开发肿瘤靶向纳米药物中的用途,可为未来应用于人骨肉瘤的靶向治疗提供基础。
[Abstract]:Osteosarcoma is a malignant bone tumor that is good for young people. The mainstream treatment at this stage is large dose chemotherapy before operation, radical resection and continuous large dose chemotherapy. Since 80s last century, the total survival rate of osteosarcoma patients is at a plateau period. The five year survival rate of primary osteosarcoma patients is about 60%-70%. The treatment of osteosarcoma is limited by the lack of targeted delivery of drugs directly to the tumor cells. Screening and identifying specific ligands for tumor cell surface receptors is a hot spot in cancer research. The emerging phage display technology is a powerful tool in this field, which may promote the development of tumor diagnosis and target therapy. Using phage display technology to screen and identify the highly affinity and specific peptide of human osteosarcoma U2OS cells, coupled with the target peptide and gold nanoscale, to evaluate the ability of peptide targeted hyperthermia osteosarcoma cells (Figure 1), to provide a basis for further development of human osteosarcoma targeted therapy. Method: 1. as a target for targeting U2OS. Cell, human osteoblast hFOB1.19 is negative subtractive cell, and Ph.D.-12 phage display library is screened for whole cell subtraction. First, the phage library and hFOB1.19 cells are fully combined to exclude the combination of hFOB1.19, that is, a partial phage of non specific binding target cells, and the remaining library is fully combined with U2OS, and the specificity is removed after washing. With a lower affinity phage, a phage with high specificity and affinity for U2OS was obtained by elution and acidification (including "cell surface binding phage" and "internalized phage"). The peptide library obtained by this round was selected for the next round of screening, and the accuracy of screening was improved after four rounds of repeated screening; 2. obtained each round. The bacteriophage coated plate was used to count the blue spot. Then the good monoclonal phage was selected randomly from the counting plate, and the sequencing company was amplified and sequenced to obtain the DNA sequence of various phage exogenous peptides. 3. the monoclonal phage of 1 of the repetition rate above was amplified, purified and then screened respectively (method synchronization sudden 1, "monoclonal phage was used. HFOB1.19, hFOB1.19, human osteosarcoma cell MG-63 and human breast cancer cell MCF-7 were selected as the control cells for ELISA detection, and the affinity and specificity of the target cells were identified by immunohistochemical staining. 5. the specificity and affinity of U2OS were selected. The high monoclonal phage is the target phage, the target polypeptide and the target polypeptide of the FITC marker are synthesized according to its DNA sequence. The target polypeptide of 6. gradient unit FITC fluorescent labeling is combined with the target cells, hFOB1.19, MG-63 and MCF-7 respectively. The affinity and specificity of target polypeptide to target cells are identified by flow cytometry, and the target polypeptide is identified as the target cells, and the target polypeptide is 7. target. After incubating the cell U2OS with the gradient target phage, the target peptides were incubated with the target peptide labeled with the quantitative FITC, and the average fluorescence intensity was detected by flow cytometry. The U2OS was incubated with the gradient target peptide and then incubated with the target phage, then the smear was used to count the blue spot. The results showed that the target phage combined with the exogenous polypeptide in the competitive combination of U2OS; 8. The target peptide was coupled with Goldnanocages (GNCs) to form a peptide gold nanoscale coupling (Peptide-GNCs, P-GNCs), and hF0B1.19 was used as the control cell. After full culture of U2OS, 808nm near infrared laser irradiation was used to record the temperature changes of cell culture fluid before and after irradiation. The cell proliferation was detected by CCK-8 method, and cell death was detected by calcine greenin staining. Results: 1. after 4 rounds of screening in vitro, the cell surface was enriched about 144 times with phage (from 1.01 x 106 PFU to 1.45 * 108pfu), and the internalized phage was enriched about 15.53 times (from 8.95 x 104pfu to 1.39 * 106 PFU); 2. randomly selected second, third and four rounds of bacteriophages after selected bacteriophages. Sequenced and named Phage OS-1, Phage OS-2 in sequence. Phage OS-404, the corresponding foreign polypeptide is named Peptide OS-1, Peptide OS-2,... Peptide OS-404, statistics of the rate of phage weight recovery, and obtained 6 high repetition rate of cell surface binding and internalized phage; 3. monoclonal phage enrichment rate experimental results show that the enrichment rate of phage is consistent with the sequence repetition rate; 4.ELISA results show that Phage OS-7 has the highest affinity for U2OS, and the affinity for MG-63 is equally high, and hFOB1.19. MCF-7 was not combined to be the target phage of this study. 5. compared with normal tissue sections of human osteosarcoma, Phage OS-7 was stained significantly positive in human osteosarcoma tissue section, while wild type phage staining results were negative; 6. peptide affinity experiment showed that PeptideOS-7 (polypeptide sequence: APWTEAYWWHLP) had the highest affinity for U2OS, and With the increase of concentration, the affinity for MG-63 was also high, and hFOB1.19 and MCF-7 were not combined. 7. phage and corresponding peptide competitive binding experiment showed that Peptide OS-7 and Phage OS-7 were competitive combined with U2OS; 8. near infrared laser irradiation experiment showed that the effect of Peptide OS-7-GNCs was obviously better than that of pure gold nanoscale group. The polypeptides we screened and identified by phage display technology are: APWTEAYWWHLP, which has high affinity and specificity to human osteosarcoma cell U2OS. This study provides a clear example of the screening and identification of U2OS specific binding peptides in human osteosarcoma cells by phage display technology, and the development of this kind of peptide in the development of human osteosarcoma cells. The use of tumor targeting nano drugs will provide a basis for future targeted therapy in human osteosarcoma.
【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R738
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