肺血管内皮细胞靶向Ang-1基因与辛伐他汀纳米共传递系统对急性肺损伤的作用及机制研究

Acute lung injury (ALI) is a disease without safe and effective therapy drugs till now, the injury of pulmonary vascular endothelial cells is the key pathogenesis of ALI, and may become a new target for drug interventions. The simvastatin nanocarriers modified with anti-ICAM-1 are found targeted to pulmonary vascular endothelial cells and lung tissue, and displayed ALI anti-inflammatory effect in our previous studies. Moreover, we found that the nanocarriers can successfully transport DNA to the nucleus and express of protein. In order to further enhance the ability of early intervention to ALI, we introduced combined drug therapy method to ALI, namely, to preparing Ang-1 gene and simvastatin combined nanocarrier in this study. The purposes are to study the physicochemical properties of the combined nanocarrier and its target ability to ALI induced pulmonary vascular endothelial cell and lung tissue. Furthermore, based on the ALI cell model and animal model successfully developed, the cellular pharmacology and animal pharmacology of the combined nanocarrier will be evaluated by determining its anti-inflammatory effect and anti apoptosis effect to ALI. Lastly, the correlation of the combined nanocarrier between PI3K / Akt signaling pathway and Ang-1 protein expression are evaluated. In all, this study aims to develop a new ALI treatment method, which combined gene therapy and chemotherapy, and from the pathogenesis of ALI, and targeted to pulmonary vascular endothelial cells.

急性肺损伤（ALI）迄今尚无安全有效的药物干预手段，肺血管内皮细胞损伤是ALI的关键发病机制，有可能成为药物干预新靶点。课题组前期构建的ICAM-1单抗辛伐他汀纳米载体具有肺血管内皮细胞和肺组织靶向性，并显示一定的ALI抗炎作用。更重要的是，我们发现纳米载体可成功输送DNA至细胞核并表达。基于此，为进一步提高ALI的早期干预能力，本项目拟通过联合用药方式，即构建Ang-1基因与辛伐他汀纳米共传递系统，研究其理化性质及靶向性，并以成功建立的ALI细胞模型和动物模型为基础，研究纳米共传递系统对ALI的细胞药效学与动物药效学，评估其抗炎作用及抗细胞凋亡作用等，最后通过探讨纳米共传递系统ALI作用与PI3K/Akt信号通路等的相关性，明确纳米共传递系统对ALI作用的关键机制。本项目旨在将基因治疗与化学药物治疗相结合，从ALI的发病机制出发，以肺血管内皮细胞为干预靶点，探寻一种ALI靶向治疗新途径。

急性肺损伤(acute lung injury，ALI)为肺炎及严重脓毒症等患者常见并发症之一，肺血管内皮细胞是ALI中最为重要的效应细胞和受损靶细胞，也是ALI药物干预的重要靶点。本研究在前期构建ICAM-1单抗辛伐他汀纳米脂质载体的基础上，以鱼精蛋白为聚阳离子， Ang-1基因为药物，通过静电吸附的原理，成功构建了ICAM-NLC/ Pro /Ang三元复合物。ICAM-NLC/ Pro /Ang三元复合物呈类球状，粒径随NLC比例增高而增大，表面带正电荷，NLC对DNA的比例大于10时，三元复合物中的DNA被完全阻滞，MTT法显示三元复合物具有良好的细胞安全性。通过LPS诱导成功构建ALI EAhy926细胞模型及小鼠动物模型， ICAM-NLC/Pro/Ang三元复合物具有经ICAM-1单抗介导的肺血管内皮细胞及ALI肺组织的靶向特性；CAM-介导的细胞内吞作用及ICAM-1单抗与ALI肺血管内皮细胞ICAM-1受体的多价相互作用是三元复合物细胞及肺组织靶向的主要作用机制。通过制备绿色荧光蛋白标记的ICAM-NLC/Pro/EGFP三元复合物，三元复合物在EAhy926模型细胞内发现ICAM-NLC/Pro/EGFP具有较高的基因转染效率。Western Blot法测定Ang-1蛋白表达结果显示，ICAM-NLC/Pro/Ang三元复合物在ALI模型细胞以及ALI模型小鼠中Ang-1蛋白表达水平，显著高于IgG-NLC/Pro/Ang组及ICAM-NLC组，表明三元复合物中的基因被成功转染，并表达Ang-1蛋白。动物药效学研究显示，ICAM-NLC /Pro/Ang（30:4:1, w/w/w）三元复合物给药后24h、48h具有显著改善小鼠BALF中TNF-α、IL-6炎症因子水平，炎症细胞浸润、肺组织病理的作用，其原因可能与 ICAM-NLC/Pro/Ang三元复合物的肺靶向作用以及Ang-1蛋白表达的血管内皮保护作用相关。本研究表明，通过基因治疗与化学治疗联合用药的方式，可改善ALI早期的炎症水平，进一步提高ALI的早期干预能力，具有潜在的临床应用价值。