肝癌微球体细胞特异性结合七肽的筛选

发布时间：2018-02-12 05:00

本文关键词： 噬菌体展示差减筛选肝癌微球体环七肽靶向载体1　出处：《贵州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:筛选能够与高转移性HCCLM3肝癌微球体特异性结合的环七肽,为后续的生物医学应用提供靶向分子。方法:利用超低吸附培养瓶和无血清培养基对HCCLM3进行培养,诱导其形成肝癌微球体。以肝癌微球体为靶细胞,HCCLM3和LO2为吸附细胞,应用噬菌体环七肽库进行全细胞差减筛选。四轮筛选后,随机挑选噬菌体单克隆进行测序;根据测序结果,采用cell-ELISA对不同类型噬菌体克隆与肝癌微球体结合的亲和力和特异性进行检测。根据特异性最高的噬菌体克隆的融合蛋白基序来合成对应的环七肽。使用竞争抑制试验检验合成多肽对噬菌体克隆与靶细胞结合的抑制作用。运用激光共聚焦显微镜进一步验证合成多肽与肝癌微球体结合的特异性。细胞毒性试验检测多肽的生物学活性。结果:通过悬浮培养,LM3细胞成功聚集成球形成微球体,四轮差减筛选后,噬菌体克隆在肝癌微球体的富集率从(8.50×10-5±1.70×10-5)%提高到(1.0×10-2±4.08×10-4)%(P0.001)。cell-ELISA结果显示P26号噬菌体克隆与肝癌微球体结合的特异性最高,其展示多肽的氨基酸序列为NTGSPYE(命名为NTG肽)。竞争抑制试验结果表明:NTG肽对P26噬菌体与肝癌微球体的结合有抑制作用(P0.01或P0.001)。运用激光共聚焦显微镜观察标记FITC的NTG肽与细胞结合的结果显示:NTG肽能与肝癌微球体特异性结合,而与无关细胞则不发生结合。细胞增殖试验显示:NTG肽对细胞生长没有增殖活性。结论:用干细胞培养体系对肝癌细胞系进行悬浮培养能稳定获得肝癌微球体。筛选到的P26号噬菌体与肝癌微球体的结合具有最高特异性,其展示的环七肽序列为NTGSPYE。合成的NTG肽具有与肝癌微球体靶向结合的能力,且对细胞的生长无影响。因此,NTG肽作为介导与肝癌干细胞特异性结合的靶向载体,在今后肝癌早期诊断和靶向治疗的应用上具有进一步开发的潜质。
[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 卤1.70 脳 10-5% to 1.0 脳 10-2 卤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.
【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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