T4噬菌体介导的靶向VEGFR-2的抗肿瘤血管生成研究

Tumor growth, invasion and metastasis are angiogenesis dependent. It has shown that, VEGF/VEGFR-2 signaling pathway plays a key role in tumor angiogenesis. Our preliminary experiments show that the recombinant bacteriophage T4 displaying of mouse vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) can specifically binds VEGF in vitro, suggesting that it may have a role in inhibiting tumor angiogenesis. In this study, a biotherapeutic method targeting VEGFR-2 will be developed by using bacteriophage T4 as the carrier. To generate recombinant phage (T4-VEGFR-2) nanoparticles, the DNA encoding the extracellular domain of VEGFR-2 will be inserted into the genome of bacteriophage T4 by homologous recombination and VEGFR-2 protein will be displayed on the T4 phage surface. Enzyme linked immunosorbent assay (ELISA), phage titration and dot hybridization methods were used to detect their ability in combination with VEGF, and to filter out the clone with strongest affinity to VEGF. Endothelial cell proliferation and migration assays will be used to detect the potential anti-tumor angiogenic activity of the recombinant phage in vitro. Tumor xenograft model and tumor metastasis model will be established and animal survival, tumor growth and metastases will be measured to evaluate the role of anti-angiogenesis of recombinant phage in vivo. In general, the aim of this study is to establish a novel anti-angiogenic strategy by using T4 phage display systerm, and to provide scientific evidence for further clinical trials of the recombinant phage (T4-VEGFR-2) nanoparticles.

恶性肿瘤的生长、侵袭和转移依赖于血管生成。VEGF/VEGFR-2信号通路在肿瘤血管生成过程中起关键作用。预实验发现，表面展示小鼠VEGFR-2的重组T4噬菌体(T4-VEGFR-2)可以特异性结合VEGF，提示其可能具有抑制肿瘤血管生成的作用。本研究以T4噬菌体作为生物纳米载体，利用同源重组的原理分别将不同长度的小鼠VEGFR-2胞外区基因插入到T4噬菌体基因组，使其表达在T4噬菌体的表面，制备成具有潜在抗肿瘤血管生成活性的重组噬菌体(T4-VEGFR-2)，酶联免疫吸附试验，噬菌体滴定，斑点杂交等方法检测其与VEGF的结合能力，并且筛选出结合能力最强的重组噬菌体，通过体外实验研究重组噬菌体对血管内皮细胞增殖和迁移的影响及其可能的作用机制；建立小鼠移植瘤模型和转移模型，观察其对荷瘤小鼠的生存期、肿瘤生长、转移的抑制作用。预期开发出一种新的抗肿瘤血管生成策略，为进一步临床实验提供科学依据。

恶性肿瘤的生长、侵袭和转移依赖于血管生成，而肿瘤血管生成的过程中VEGF/VEGFR-2信号通路起关键作用。因此，通过阻断VEGF/VEGFR-2信号通路可以探索一种新的抗肿瘤治疗方法。本研究我们采用T4噬菌体展示技术，将小鼠VEGFR-2展示于T4噬菌体的表面，制备成重组噬菌体T4-VEGFR-2，然后研究其抗肿瘤效应。结果发现：VEGFR2-1-4、VEGFR2-1-5、VEGFR2-1-6 和VEGFR2-1-7结构域均成功展示于噬菌体的表面，酶联免疫吸附-噬菌体滴定和噬菌体-酶联免疫吸附试验证实含有不同结构域的噬菌体均可以结合VEGF。经体外实验证实，重组噬菌体T4-VEGFR-2可以抑制VEGF诱导的血管内皮细胞增殖和迁移。体内实验室证实，T4-VEGFR-2可以抑制Lewis肺癌和结肠癌皮下移植瘤生长，并且可以延长小鼠生存时间。免疫组化显示，重组噬菌体T4-VEGFR-2可以减少肿瘤微血管密度。本研究开发出一种新的肿瘤抗血管生成策略，为进一步临床试验提供了科学依据。依托本项目，已经发表1篇论文，并且被SCI收录，1篇已经完成撰写，准备投稿，已经申请2项发明专利。另外，还培养了3名硕士研究生。