表皮生长因子受体介导的靶向性基因治疗导入系统

发布时间：2018-06-28 05:17

  本文选题：GE11 + 靶向性噬菌粒颗粒　； 参考：《复旦大学》2005年博士论文

【摘要】：肿瘤基因治疗有望成为除手术、放疗和化疗等方法外的又一新的抗癌策略。尤其在抗肿瘤转移和复发方面将起重要作用。目前还缺乏将基因导入肿瘤细胞的高效靶向性载体系统,这是肿瘤基因治疗至今尚未成为临床常规治疗措施的关键因素之一。表皮生长因子受体(EGFR)由于在很多肿瘤有高表达,因此是一个很好的靶点。本研究旨在建立一种靶向EGFR受体的基因导入系统并检验其有效性,为肿瘤的靶向性基因治疗提供可靠的理论依据与实践指导。 第一部分 利用噬菌体多肽展示文库筛选表皮生长因子受体的配体多肽 【目的】寻求可以与表皮生长因子受体(EGFR)受体特异性结合的配体多肽。【方法】以EGFR蛋白为靶,利用噬菌体展示随机12肽库进行筛选;或者构建稳定高表达EGFR突变体Ⅲ(EGFRvⅢ)胞外区的U87细胞系,以该细胞系为筛选靶,利用环7肽库和12肽库进行筛选。经过多轮筛选后,随机挑选克隆进行测序。把富集的克隆通过免疫组化及噬菌体回收试验鉴定获得可以与靶蛋白结合的克隆;进一步采用内吞实验研究噬菌体阳性克隆的特征。【结果】通过EGFR蛋白为靶筛选,经过三轮筛选获得展示GE11(YHWYGYTPQNVI)多肽的噬菌体Phage-GE11。噬菌体回收实验中,Phage-GE11可以和EGFR有效结合,而且可以被EGF和GE11多肽所竞争抑制。免疫组化结果显示Phage-GE11可以和EGFR表达阳性的细胞SMMC-7721,NCI-H460结合,但和EGFR表达阴性的细胞K-562细胞没有结合。此外,免疫荧光结果显示Phage-GE11可以被SMMC-7721细胞所内吞。但是,以高表达EGFR突变体Ⅲ的细胞系作为筛选靶进行筛选并没有获得阳性克隆。【结论】噬菌体文库展示技术是一项获得配体多肽的有效技术,特别是以纯化蛋白作为筛选靶可以更有效地获得阳性克隆。Phage-GE11可以与EGFR特异结合,并被EGFR表达阳性的细胞所内吞。
[Abstract]:Tumor gene therapy is expected to be a new anticancer strategy in addition to surgery, radiotherapy and chemotherapy. Especially in anti-tumor metastasis and recurrence will play an important role. At present, there is still a lack of efficient targeting vector system for gene transfer into tumor cells, which is one of the key factors of tumor gene therapy. Epidermal growth factor receptor (EGFR) is a good target because of its high expression in many tumors. The purpose of this study is to establish an EGFR receptor targeting gene transfer system and to test its effectiveness, and to provide a reliable theoretical basis and practical guidance for tumor targeting gene therapy. The first part is to screen the ligand peptide of epidermal growth factor receptor (EGF receptor) by phage display library [objective] to find out whether EGF receptor can be associated with epidermal growth factor receptor (EGF). (EGFR) receptor-specific ligand peptide. [methods] EGFR protein was used as target, Using phage display random 12 peptide library to screen, or to construct U87 cell line with high expression of EGFR mutant 鈪，

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