超声微泡负载的多靶点药物靶向阻断胃癌Notch通路和抑制新生血管形成的实验研究

Angiopoiesis has been proved to be correlated with tumorigenesis and metastasis of gastric cancer, a common malinant tumor in China, so demonstrating the mechanism of tumor angiopoiesis and exploring new signalling pathway drugs target angiopoiesis are urgent for gastric cancer research. This study is motivated by previous research achievement on the the Notch signalling as a key pathway of tumor vasculature. Our first work is to demonstrate the mechanism of Notch signalling pathway on gastric cancer angiogenesis by RT-qPCR, western blot and immunehistochemical stainning. The second is to fabricate a DLL4 targeted lipid ultrasound microbubble with biotin-avidin system employed as bridging molecule, and to investgate the physical properties and targeting functionin of the ultrasound microbubble. Finally, the nude mice model of gastric cancer is established with human gastric cancer cell line, and treated via blockage of DLL4/Notch pathway with microbubble-mediated DLL4 antibody and γ-secretase inhibitor. To evaluate the suppression of the intervention strategies and clairify its inhibiting mechanism, MicroPET is used to dynamically monitor the growth of the gastric cancer, combined with genetic and molecular biological detection. Our research result will be of great significance to provide both theoretical and technological basis for a safe molecular-targeted therapy of gastric cancer.

胃癌是我国常见的恶性肿瘤之一，其发展和转移都与新生血管形成密切相关，探索能特异、高效阻断胃癌新生血管形成的新基因和信号通路靶点是胃癌和肿瘤学研究的新方向。目前应用的新型抗血管形成靶向分子药物仍存在肿瘤拮抗和并发症发生率高的问题。本项目基于前期Notch信号通路对胃癌新生血管发生影响的研究成果，运用基因、蛋白和免疫组化等技术手段明确DLL4抗体和γ-分泌酶抑制剂在体外细胞模型上对胃癌Notch通路的协同阻断作用。以分子影像技术为基础，制备结合DLL4抗体的超声微泡造影剂，考量其理化性能、成像性能及靶向性能，利用超声微泡介导的DLL4抗体和γ-分泌酶抑制剂多靶点阻断Notch通路，应用MicroPET动态跟踪胃癌生长状况，在大体和分子水平上阐明并评价联合干预措施抑制胃癌生长的作用机制和影响规律，从而为胃癌的分子靶向治疗提供可靠的理论依据和技术基础。

探索能特异、高效抑制胃癌恶性生物学行为的信号通路靶点是胃癌和肿瘤学研究的新方向。本项目基于前期Notch信号通路有关研究进展，运用基因、蛋白和免疫组化等技术手段明确DLL4抗体和γ-分泌酶抑制剂在体外细胞模型上对胃癌Notch通路的协同阻断作用。同时，以分子影像技术为基础，制备结合DLL4抗体的超声微泡造影剂，利用超声微泡介导的DLL4抗体和γ-分泌酶抑制剂多靶点阻断Notch通路，探索并评价联合干预措施抑制胃癌生长的有效性，希望为胃癌的分子靶向治疗提供可靠的技术基础。我们首先在胃癌细胞系及临床标本中验证了DLL4与Notch受体的表达，并证实antiDLL4及DAPT可以有效的协同抑制胃癌细胞的恶性生物学行为。随后，我们制备了靶向anti-DLL4的超声微泡，并利用超声靶向微泡运送技术在胃癌动物模型体内证实了antiDLL4及DAPT联合用药可以有效抑制胃癌生长转移等恶性行为，并有效诱导了凋亡相关蛋白在肿瘤细胞的表达。我们的实验不仅证实了anti-DLL4和DAPT具有协同抑制胃癌的作用，更为开展进一步的临床转化研究提供了坚实的实验室依据。