基于间充质干细胞的靶向泡状棘球蚴组织抗血管新生基因治疗的实验研究

Alveolar Echinococcosis (AE) is a potentially fatal zoonosis caused by Echinococcus multilocularis (E.m), showing the growth of malignant invasion, can secondary to lung and brain metastasis, and surgical and medical therapeutic efficacy is not good. Similar to malignant tumor, angiogenesis is a critical process for biological behavior of the parasite and it may be the effective way to control the parasite invasion and metastasis by inhibiting local angiogenesis. Thus, the key point is how to precisely inhibit the local angiogenesis of the alveolar echinococcus invaded tissue without damage the host. It has been already known that mesenchymal stem cells (MSCs) can serve as a vehicle to transport a therapy gene to tumor target, which is the currently focus in the study of tumor accurate treatment. Similarly, we also found that recruitment of MSCs to the hepatic alveolar echinococcus tissue after intravenous injection. Therefore, we propose research hypotheses: to use MSCs transport Endostatin gene for the Alveolar Echinococcosis therapy. In this study, we plan to elucidate the molecular mechanism of MSCs migrate to the hepatic alveolar echinococcus tissue by using flow cytometry, magnetic activated cell sorting, pathology organization analysis, and the living cell fluorescence trace techniques. Moreover, Endostatin gene recombinant MSCs will be applied as the therapeutic gene carrier in treatment of the AE animal model to evaluate the therapeutic efficacy including the changes of local angiogenesis inhibition and the survival of the animal model. The study can not only reveal the mechanism of migration of stem cells to microenvironment of heterogeneity but also provide a new way to AE gene therapy and offer a new opportunity to address unmet medical need in AE patient population.

泡状棘球蚴病是一种危害严重的人畜共患寄生虫病，呈浸润性生长并可转移，手术与药物治疗效果均不理想。与恶性肿瘤相似，血管新生亦是泡状棘球蚴生物学行为的重要特征，抑制局部血管新生可能抑制泡状棘球蚴的生长和转移。如何在不影响宿主的前提下，精准抑制泡状棘球蚴组织局部血管新生是关键。应用干细胞携带治疗基因能够精确定位肿瘤组织，是当前肿瘤精准治疗研究的热点。预实验显示：静脉输送的间充质干细胞最终聚集定位于小鼠模型肝泡状棘球蚴组织。因此，提出假说：基于间充质干细胞的靶向泡状棘球蚴组织抗血管新生基因治疗。本研究拟通过一系列技术手段，探讨间充质干细胞定向泡球蚴迁移分子机制。并在此基础上，结合内皮抑制素的抑血管新生作用，研究间充质干细胞靶向泡球蚴基因治疗可行性，以及对泡球蚴小鼠生存状态的影响。研究结果不仅补充干细胞定向异种组织迁移的机制，而且为泡状棘球蚴病的精准治疗提供一个新的思路。

泡状棘球蚴病是一种危害严重的人畜共患寄生虫病，呈浸润性生长并可转移，手术与药物治疗效果均不理想。与恶性肿瘤相似，血管新生亦是泡状棘球蚴生物学行为的重要特征，抑制局部血管新生可能抑制泡状棘球蚴的生长和转移。如何精准抑制泡状棘球蚴组织局部血管新生是关键。本研究探讨间充质干细胞定向泡状棘球蚴迁移分子机制。并在此基础上，结合内皮抑制素的抑血管新生作用，研究间充质干细胞靶向泡状棘球蚴基因治疗可行性，以及对泡状棘球蚴小鼠生存状态的影响。研究发现：泡状棘球蚴囊肿生长与其血管系统的建立呈相互促进的作用；泡状棘球蚴与宿主之间相互作用集中体现在二者相接触的炎性反应带中，血管生成及其相关因子分泌表达均由该区域细胞参与实现；VEGFA在泡状棘球蚴感染后持续高水平表达，并能进入循环系统对全身产生作用；其中VEGFR2的表达及磷酸化水平升高，受 VEGFA升高的调控最显著；HIF-1α抑制剂能够抑制VEGFA的表达；VEGF抑制剂能够抑制小鼠泡状棘球蚴组织血管生成，阻碍泡状棘球蚴生发层的形成，抑制囊肿增生；泡状棘球蚴组织感染后NF-κB、SDF-1α、VCAM-1水平升高，NF-κB抑制剂能够抑制SDF-1α、VCAM-1的表达，通过抑制血管内皮细胞的迁移抑制血管生成，抑制泡状棘球蚴囊肿生长；MSCs表达VEGFR2，CXCR4以及VLA-4，能够通过VEGFA/ VEGFR2生物轴，SDF-1α/CXCR4生物轴，VCAM-1/VLA-4生物轴，靶向泡状棘球蚴组织迁移并定殖。ES-MSCs表达基因重组内皮抑素，其分化能力，表面标记，以及定向泡状棘球蚴组织迁移能力与WT MSC相比未发生明显改变； ES-MSC治疗肝泡状棘球蚴小鼠，ES-MSCs定殖泡状棘球蚴组织，分泌内皮抑素，通过抑制VEGFR2磷酸化，抑制泡状棘球蚴组织血管生成，实现抑制泡状棘球蚴囊肿生长；此外，MSCs可能通过下调CD8＋T发挥抗泡状棘球蚴感染免疫调节作用。研究结果不仅补充间充质干细胞定向异种组织迁移的机制，而且为泡状棘球蚴病的精准治疗提供一个新的思路。