本文關(guān)鍵詞： 噬菌體展示 差減篩選 肝癌 微球體 環(huán)七肽 靶向載體1　出處：《貴州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:篩選能夠與高轉(zhuǎn)移性HCCLM3肝癌微球體特異性結(jié)合的環(huán)七肽,為后續(xù)的生物醫(yī)學(xué)應(yīng)用提供靶向分子。方法:利用超低吸附培養(yǎng)瓶和無血清培養(yǎng)基對(duì)HCCLM3進(jìn)行培養(yǎng),誘導(dǎo)其形成肝癌微球體。以肝癌微球體為靶細(xì)胞,HCCLM3和LO2為吸附細(xì)胞,應(yīng)用噬菌體環(huán)七肽庫進(jìn)行全細(xì)胞差減篩選。四輪篩選后,隨機(jī)挑選噬菌體單克隆進(jìn)行測(cè)序;根據(jù)測(cè)序結(jié)果,采用cell-ELISA對(duì)不同類型噬菌體克隆與肝癌微球體結(jié)合的親和力和特異性進(jìn)行檢測(cè)。根據(jù)特異性最高的噬菌體克隆的融合蛋白基序來合成對(duì)應(yīng)的環(huán)七肽。使用競(jìng)爭(zhēng)抑制試驗(yàn)檢驗(yàn)合成多肽對(duì)噬菌體克隆與靶細(xì)胞結(jié)合的抑制作用。運(yùn)用激光共聚焦顯微鏡進(jìn)一步驗(yàn)證合成多肽與肝癌微球體結(jié)合的特異性。細(xì)胞毒性試驗(yàn)檢測(cè)多肽的生物學(xué)活性。結(jié)果:通過懸浮培養(yǎng),LM3細(xì)胞成功聚集成球形成微球體,四輪差減篩選后,噬菌體克隆在肝癌微球體的富集率從(8.50×10-5±1.70×10-5)%提高到(1.0×10-2±4.08×10-4)%(P0.001)。cell-ELISA結(jié)果顯示P26號(hào)噬菌體克隆與肝癌微球體結(jié)合的特異性最高,其展示多肽的氨基酸序列為NTGSPYE(命名為NTG肽)。競(jìng)爭(zhēng)抑制試驗(yàn)結(jié)果表明:NTG肽對(duì)P26噬菌體與肝癌微球體的結(jié)合有抑制作用(P0.01或P0.001)。運(yùn)用激光共聚焦顯微鏡觀察標(biāo)記FITC的NTG肽與細(xì)胞結(jié)合的結(jié)果顯示:NTG肽能與肝癌微球體特異性結(jié)合,而與無關(guān)細(xì)胞則不發(fā)生結(jié)合。細(xì)胞增殖試驗(yàn)顯示:NTG肽對(duì)細(xì)胞生長沒有增殖活性。結(jié)論:用干細(xì)胞培養(yǎng)體系對(duì)肝癌細(xì)胞系進(jìn)行懸浮培養(yǎng)能穩(wěn)定獲得肝癌微球體。篩選到的P26號(hào)噬菌體與肝癌微球體的結(jié)合具有最高特異性,其展示的環(huán)七肽序列為NTGSPYE。合成的NTG肽具有與肝癌微球體靶向結(jié)合的能力,且對(duì)細(xì)胞的生長無影響。因此,NTG肽作為介導(dǎo)與肝癌干細(xì)胞特異性結(jié)合的靶向載體,在今后肝癌早期診斷和靶向治療的應(yīng)用上具有進(jìn)一步開發(fā)的潛質(zhì)。
[Abstract]:Objective: to screen the cyclopeptide which can specifically bind to microspheres of HCCLM3 liver cancer with high metastasis, and to provide targeted molecules for further biomedical application. Methods: HCCLM3 was cultured in ultra-low adsorption culture flask and serum-free medium. HCCLM3 and LO2 were used as adsorption cells, and phage cyclopeptide library was used for whole cell subtractive screening. After four rounds of screening, phage phage monoclonal was randomly selected for sequencing. Cell-ELISA was used to detect the affinity and specificity of different types of phage clones combined with hepatoma microspheres. The corresponding cyclic heptapeptide was synthesized according to the fusion protein motif of bacteriophage clone with the highest specificity. Competitive inhibition was used. The inhibitory effect of synthetic polypeptide on phage clone and target cell binding was tested. The specificity of the binding of synthetic polypeptide to hepatoma microsphere was further verified by laser confocal microscopy. Cytotoxicity test was used to detect the bioactivity of polypeptide. Results: through suspension culture, LM3 cells were successfully assembled into spheres to form microspheres. After four rounds of differential subtractive screening, the enrichment rate of P26 phage clone in hepatoma microspheres increased from 8.50 脳 10-5 鹵1.70 脳 10-5% to 1.0 脳 10-2 鹵4.08 脳 10-4. Cell-ELISA showed that P26 phage clone combined with hepatoma microspheres with the highest specificity. The amino acid sequence of the displayed peptide was NTGSPYE (named NTG peptide). The results of competitive inhibition test showed that the binding of P26 phage to hepatoma microsphere could be inhibited by the peptide. The NTG labeled with FITC was observed by laser confocal microscopy. The binding of peptide to cell showed that the peptide of 1% NTG could specifically bind to microsphere of liver cancer. The cell proliferation test showed that the cell growth activity of the peptide was not proliferative. Conclusion: the suspension culture of hepatoma cell line with stem cell culture system can stably obtain the microsphere of liver cancer. The binding of P26 phage to hepatoma microsphere has the highest specificity. The synthesized NTG peptide has the ability to target the hepatoma microspheres and has no effect on the growth of the cells. Therefore, NTG peptide is used as a targeting vector to mediate the specific binding with hepatoma stem cells. It has potential for further development in early diagnosis and targeted therapy of liver cancer.
【學(xué)位授予單位】：貴州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 Wanqing Chen;Rongshou Zheng;Hongmei Zeng;Siwei Zhang;Jie He;;Annual report on status of cancer in China, 2011[J];Chinese Journal of Cancer Research;2015年01期

2 Abeer A Bahnassy;Abdel-Rahman N Zekri;Ahmed El-Bastawisy;Amal Fawzy;Marwa Shetta;Nehal Hussein;Dalia Omran;Abdallah A S Ahmed;Samir S El-Labbody;;Circulating tumor and cancer stem cells in hepatitis C virusassociated liver disease[J];World Journal of Gastroenterology;2014年48期

3 王寒琪;王晉星一;陳林;陳妮;陳我嬋;潘婭;洪陽;陳騰祥;;不同遷移能力肝癌細(xì)胞中干細(xì)胞相關(guān)基因Nanog、Sox2和Oct4的表達(dá)[J];貴陽醫(yī)學(xué)院學(xué)報(bào);2013年03期

4 Arun Budha;Thomas D Wang;;Affinity peptide developed by phage display selection for targeting gastric cancer[J];World Journal of Gastroenterology;2012年17期

5 梁晨;單娟娟;沈俊杰;劉立;錢程;;肝癌細(xì)胞系Huh7中腫瘤干細(xì)胞分離及鑒定[J];浙江理工大學(xué)學(xué)報(bào);2011年04期

6 陳孝平,裘法祖,吳在德,何松青,張萬廣,劉安重,孟春城,江斌,喬森,潘華鋒,史光軍,曹斌;肝細(xì)胞癌門靜脈癌栓形成的分子生物學(xué)機(jī)制研究[J];中華實(shí)驗(yàn)外科雜志;2005年09期

，

本文編號(hào)：1504835