本文選題：骨肉瘤 + 噬菌體文庫��； 參考：《廣州中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：骨肉瘤是一種好發(fā)于青少年的惡性骨腫瘤。現(xiàn)階段主流的治療手段是術(shù)前大劑量化療、根治性切除及術(shù)后繼續(xù)大劑量化療。自上世紀(jì)八十年代以來,骨肉瘤患者總生存率長期處于平臺期,原發(fā)性骨肉瘤患者五年生存率約為60%-70%。現(xiàn)階段骨肉瘤的治療受限于缺乏能直接將藥物遞送到腫瘤細(xì)胞的靶向載體。篩選與鑒別腫瘤細(xì)胞表面受體的特異性配體,是目前腫瘤研究的熱點,新興的噬菌體展示技術(shù)是這一領(lǐng)域的強(qiáng)大工具,將可能促進(jìn)腫瘤診斷和靶向治療的發(fā)展。目的:利用噬菌體展示技術(shù)篩選并鑒定對人骨肉瘤U2OS細(xì)胞具有高度親和力和特異性的多肽,進(jìn)一步將靶向多肽與金納米籠偶聯(lián),評價多肽靶向熱療骨肉瘤細(xì)胞的能力(圖1),為進(jìn)一步開發(fā)人骨肉瘤的靶向治療提供基礎(chǔ)。方法:1.以U2OS作為靶細(xì)胞,人成骨細(xì)胞hFOB1.19為陰性消減細(xì)胞,對Ph.D.-12噬菌體展示文庫進(jìn)行全細(xì)胞差減篩選。首先將噬菌體文庫與hFOB1.19細(xì)胞充分結(jié)合,排除與hFOB1.19結(jié)合,即非特異性結(jié)合靶細(xì)胞的部分噬菌體,將剩下的文庫與U2OS充分結(jié)合,洗滌后去除特異性與親和力較低的噬菌體,通過洗脫及酸化裂解得到對U2OS具有較高特異性和親和力的噬菌體(包括"細(xì)胞表面結(jié)合噬菌體"及"內(nèi)化噬菌體"),將這一輪得到的肽庫投入下一輪的篩選,經(jīng)過四輪重復(fù)篩選提高篩選的精確性;2.將每一輪得到的噬菌體涂板進(jìn)行藍(lán)斑計數(shù),隨后從計數(shù)平板中隨機(jī)挑選分離良好的單克隆噬菌體,擴(kuò)增后送測序公司進(jìn)行序列測定,獲得各噬菌體外源多肽的DNA序列;3.將上述重復(fù)率1的單克隆噬菌體擴(kuò)增、純化,再分別投入篩選(方法同步驟1,"單克隆噬菌體"代替"噬菌體文庫")進(jìn)行反向鑒定;4.挑選重復(fù)率較高的單克隆噬菌體,以hFOB1.19、人骨肉瘤細(xì)胞MG-63及人乳腺癌細(xì)胞MCF-7為對照細(xì)胞進(jìn)行ELISA檢測、免疫組織化學(xué)染色鑒定其對靶細(xì)胞的親和力及特異性;5.挑選與U2OS特異性及親和力最高的單克隆噬菌體為目標(biāo)噬菌體,依據(jù)其DNA序列合成目標(biāo)多肽和FITC標(biāo)記的目標(biāo)多肽;6.梯度單位FITC熒光標(biāo)記的目標(biāo)多肽分別與靶細(xì)胞、hFOB1.19、MG-63及MCF-7結(jié)合,通過流式細(xì)胞儀檢測熒光值鑒定目標(biāo)多肽對靶細(xì)胞的親和力及特異性;7.靶細(xì)胞U2OS與梯度目標(biāo)噬菌體孵育后,與定量FITC標(biāo)記的目標(biāo)多肽孵育,流式細(xì)胞儀檢測平均熒光強(qiáng)度;U2OS與梯度目標(biāo)多肽孵育,然后與目標(biāo)噬菌體孵育,隨后涂板進(jìn)行藍(lán)斑計數(shù),綜合以上結(jié)果驗證目標(biāo)噬菌體與其外源多肽競爭性結(jié)合U2OS;8.將目標(biāo)多肽與金納米籠(Goldnanocages,GNCs)偶聯(lián)形成多肽-金納米籠偶聯(lián)物(Peptide-GNCs,P-GNCs),以hF0B1.19為對照細(xì)胞,與U2OS充分培養(yǎng)后,808nm近紅外激光照射,記錄照射前后細(xì)胞培養(yǎng)液溫度變化,CCK-8法檢測細(xì)胞增殖、鈣黃綠素染色檢測細(xì)胞死亡情況。結(jié)果:1.經(jīng)過4輪體外篩選后,細(xì)胞表面結(jié)合噬菌體富集了約144倍(由1.01× 106 pfu增長至1.45×108pfu),內(nèi)化噬菌體富集了約15.53倍(由8.95×104pfu增加至1.39×106 pfu);2.分別隨機(jī)挑選第二、三、四輪篩選后的噬菌體81、123、200個,共404個噬菌體進(jìn)行測序,并按順序命名為Phage OS-1、Phage OS-2、…Phage OS-404,相應(yīng)外源多肽命名為Peptide OS-1、Peptide OS-2,…Peptide OS-404,統(tǒng)計各噬菌體重復(fù)率,得到重復(fù)率較高的細(xì)胞表面結(jié)合及內(nèi)化噬菌體各6個;3.單克隆噬菌體富集率實驗結(jié)果表明:各噬菌體富集率與測序重復(fù)率相符;4.ELISA結(jié)果顯示:Phage OS-7對U2OS親和力最高,對MG-63親和力同樣較高,與hFOB1.19、MCF-7不結(jié)合,為本次研究的目標(biāo)噬菌體;5.與人骨肉瘤癌旁正常組織切片對比,Phage OS-7在人骨肉瘤組織切片染色呈顯著陽性,而野生型噬菌體染色結(jié)果為陰性;6.多肽親和力實驗顯示:PeptideOS-7(多肽序列為:APWTEAYWWHLP)對U2OS親和力最高,且隨著濃度的增加而升高,對MG-63親和力同樣較高,對hFOB1.19、MCF-7不結(jié)合;7.噬菌體及相應(yīng)多肽競爭性結(jié)合實驗表明:Peptide OS-7與Phage OS-7競爭性結(jié)合U2OS;8.近紅外激光照射實驗顯示:Peptide OS-7-GNCs的光熱治療效果明顯優(yōu)于單純金納米籠組。結(jié)論:我們通過噬菌體展示技術(shù)篩選及鑒定的多肽序列為:APWTEAYWWHLP,該多肽對人骨肉瘤細(xì)胞U2OS具有高度親和力和特異性。本研究提供了通過噬菌體展示技術(shù)篩選及鑒定人骨肉瘤細(xì)胞U2OS特異性結(jié)合多肽的明確實例,以及此類多肽在開發(fā)腫瘤靶向納米藥物中的用途,可為未來應(yīng)用于人骨肉瘤的靶向治療提供基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣州中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R738
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本文編號：1951664