稳定表达双特异性免疫毒素的骨髓间充质干细胞靶向杀伤胶质瘤血管及机制研究

As is known to all that angiogenesis plays a key role in growth of malignant gliomas,and blocking the blood supply to glioma is the most promising treatment strategies. We have demonstrated hMSC may serve as an effective platform for the targeted delivery of immunotoxin and VEGF-PE38 secreting hMSCs was effective at killing endothelial cells in a malignant glioma tumor model. However, recent studies have reported a new tumor microcirculation pattern, vasculogenic mimicry (VM), in malignant glioma and other tumors which participate in tumor blood supply accompanied by endothelium-dependent blood vessels. Therapeutic strategies that target endothelial cells have no effect on tumor cells that engage in VM. Novel strategies both killing endothelium-dependent blood vessels and VM channels are in great demand.Further studies have found that the EphA2 receptor is overexpressed on tumor cells and plays an key role duiring VM. With this in mind, we hypothesize that dua-specific immunotoxin VEGF-EphrinA1-PE38 could target both endothelial cells and tumor cells that engage in VM. For this project, hMSCs were genetically manipulated with an lentival vector encoding VEGF-EphrinA1-PE38. resulting in higher efficiency and long-term expression of the novel dual-specific immunotoxin. The ex vivo and in vivo studies will confirmed the therapeutic efficacy and reveal the mechanism of killing both endothelium-dependent blood vessels and VM channels. providing a novel strategy of anti-angiogenesis therapy for malignant gliomas.

血管生成在胶质瘤浸润生长过程中起关键作用，阻断肿瘤血供是最具潜力的治疗策略。我们已经证明间充质干细胞可以作为表达免疫毒素的载体细胞，分泌重组免疫毒素VEGF-PE38的骨髓间充质干细胞能够特异性杀伤胶质瘤内皮血管。然而，研究表明恶性胶质瘤血管中不仅存在内皮血管，同时还存在拟态血管。只有对肿瘤内皮血管和拟态血管双重杀伤，才能较彻底地阻断血供从而遏制肿瘤生长。进一步体外研究证明，参与形成拟态血管的胶质瘤细胞表面EphA2受体特异性高度表达。由此，我们首次提出双特异性免疫毒素VEGF-EphrinA1-PE38同时阻断胶质瘤内皮血管和拟态血管的可能性。本项目拟构建稳定表达该双特异性免疫毒素的骨髓间充质干细胞，体内移植后观察是否对内皮血管和拟态血管具有双重杀伤效应，旨在获得该双价免疫毒素特异性靶向杀伤内皮血管和拟态血管的可靠证据及作用机制，探索胶质瘤抗血管治疗的新策略。

目的：构建同时针对肿瘤内皮血管和拟态血管的重组免疫毒素VEGF165- EphrinA1-PE38KDEL，利用骨髓间充质干细胞作为载体，观察其对脑胶质瘤的治疗作用。.方法：采用DNA克隆和突变技术构建双靶向重组免疫毒素VEGF165- EphrinA1-PE38KDEL的慢病毒载体，再通过慢病毒系统转染骨髓间充质干细胞，从基因及蛋白水平验证转染后骨髓间充质干细胞对重组免疫毒素的表达，通过体外实验观察其对胶质瘤细胞的选择性杀伤作用，然后移植入U87胶质瘤细胞裸鼠移原位植瘤模型，观察分泌重组免疫毒素的骨髓基质干细胞对脑胶质瘤的治疗作用。..结果：成功建立了可持续分泌重组免疫毒素VEGF165-EphrinA1-PE38KDEL的骨髓充质干细胞。体外细胞毒性实验表明，hMSCs分泌的重组免疫毒素VEGF165- EphrinA1-PE38KDE对胶质瘤细胞及内皮细胞均具有体外杀伤效应，降低内皮细胞及胶质瘤细胞的体外管道形成能力。抗体中和实验表明VEGF165-EphrinA1-PE38KDEL具有特异性靶向杀伤效应。瘤内注射重组免疫毒素转染的hMSCs，体内实验发现瘤内注射免疫毒素转染的细胞能够显著抑制胶质瘤生长，双特异性免疫毒素较单靶免疫毒素对胶质瘤荷瘤小鼠有更明显的其抗肿瘤效应，免疫毒素VEGF165-PE38KDEL治疗后肿瘤组织VM增高。双靶免疫毒素VEGF165-EphrinA1-PE38KDEL能阻断胶质瘤微血管密度及拟态血管；其抗肿瘤效应与免疫毒素杀伤肿瘤内皮细胞及拟态血管有关。. 结论：本研究以重组免疫毒素为治疗策略，靶向胶质瘤新生内皮血管及拟态血管，旨在通过阻断肿瘤血管从而抑制肿瘤，利用hMSCs肿瘤趋向性等特点，以hMSCs作为基因治疗的载体，以VEGFR和EphA2为靶点，构建了分泌双靶重组免疫毒素VEGF165-EphrinA1-PE38KDEL的hMSCs，对其体内外靶向抗胶质瘤作用进行了初步评价，通过免疫组化，分析重组免疫毒素治疗对肿瘤微血管密度和拟态血管的影响，获得了双靶向免疫毒素可对胶质瘤内皮血管及拟态血管同时杀伤的依据，进一步验证了hMSCs可作为基因治疗载体细胞的可行性，在国内外尚未见报道，为胶质瘤的抗血管治疗和免疫毒素靶向治疗研究进行了一些有益的探索。