葡萄糖基修饰pH敏感脂质体包被二氯乙酸盐靶向肺血管重构的实验研究

PAH is one of insidious but fatal pan-vasculopathy diseases. The degenerative status of distal pulmonary vessels co-exists with the proliferating neointimal formation in pre-capillary arterioles in patients with PAH at the advanced stage. Our previous study indicated that early intervention with dichloroacetate can effectively prevent the neointimal formation, but late intervention was incapability in reversal of proliferating intimal lesions in rats treated with monocrotaline following left pneumonectomy. The reasons can be as follows: firstly, the narrowing or occlusion of pulmonary arteriole lumens caused by neointimal lesions precludes the distribution of DCA in these injured intraacinar or preacinar vessels. Secondly, in addition to pro-apoptosis effect of DCA on proliferating intimal lesions, DCA also has detrimental effect on normal or apoptotic endothelial cells in pulmonary microvessels, which aggravates the endothelial dysfunction and leads to pulmonary remodeling. There is considerable evidence supporting that several key factors related to cellular glycolysis including HIF-1α, hexokinase-2, glucose transport-1 have been shown markedly up-regulated in the areas of intimal lesions. The glycolytic status even under normoxia of pulmonary arterial smooth muscle cells due to mitochondria dysfunction is accompanied with hyperpolarization of mitochondria membrane potential and cell apoptosis-resistance. Based on these aforementioned findings, therefore, dichloroacetate loaded glucose-modified pH sensitive liposomes were constructed in this study and the in-vitro study was applied to testify the targeting character and drug releasing efficiency at pH (6.5~7.0) in those liposomes. The biodistribution of liposomes were identified by in vivo and ex vivo nearinfrared fluorescence imaging and further analyzed quantitatively by high performance liquid chromatography and the targeted conjunction of liposomes with neointimal lesion areas in rats was evaluated with confocal laser scanning microscopy. Finally the selective pro-apoptosis effect of liposomes on the proliferation of neointimal formation and the underlying mechanism were also investigated.

肺动脉高压发病隐匿，确诊时患者远端肺小血管退行性病变和内膜增殖病变并存。我们前期的研究证实：二氯乙酸盐(DCA) 干预对肺高压大鼠的内膜增殖没有明显作用，可能与新生内膜造成管腔狭窄，影响了DCA在目标部位的分布；DCA对处于正常或者凋亡状态的内皮细胞的促凋亡作用恶化了内皮功能障碍等因素有关。本课题我们针对肺血管内膜增殖性损伤部位HIF-1α，己糖激酶-2（HXK-2）、葡萄糖转运体-1（ GLUT-1）高表达，糖酵解增强的特征，设计相应的靶向葡萄糖头基与pH敏感脂质体连接，通过体外实验筛选出1~2个稳定的、对pH(6.5~7.0)敏感的靶向脂质体。将DCA包封在脂质体中静脉注射，通过活体示踪、高效液相色谱、免疫荧光测定DCA在大鼠不同组织分布，及对新生内膜的靶向结合能力，观察其选择性对肺血管增殖内膜部位的促凋亡作用以及对HIF-1α、HXK-2等蛋白的影响,为治疗晚期肺高压提供新的思路。

肺动脉高压是以肺血管阻力增加，原位血栓形成，肺血管重构为特征的一组肺血管损伤性疾病。肺动脉平滑肌细胞过度增殖和凋亡下降是血管重构的主要病理改变。如何靶向增殖的肺动脉平滑肌细胞，抑制其过度生长、促进其凋亡是逆转肺血管重构的关键。肺小动脉平滑肌细胞表现为线粒体能量代谢的异常，有氧条件下糖酵解作用增强。临床和基础研究发现：肺高压来源的肺血管平滑肌细胞葡萄糖转运体-1（GLUT-1）上调，葡萄糖摄取增加、己糖激酶活性增强，线粒体膜电势超极化，线粒体相关凋亡途径抑制。西地那非作为磷酸二酯酶5选择性抑制剂，能升高细胞内cGMP含量，激活PKG信号通路，促进平滑肌细胞舒张和抑制细胞增殖，迁徙，促进凋亡。但是它生物利用度低，半衰期短，还不能明显延长肺高压患者的寿命。我们的研究表明：（1）野百合碱肺高压大鼠模型上大鼠肺血管GLUT-1表达上调，肺组织和右心室5-FDG摄取增加，（2）我们构建了葡萄糖基修饰的脂质体，旨在通过GLUT-1，提高肺动脉平滑肌细胞对葡萄糖基修饰脂质体的摄取。我们的体外实验表明，肺动脉平滑肌细胞摄取脂质体具有剂量和时间依赖性；相比普通脂质体，葡萄糖基修饰的脂质体静脉注射后具有肺组织靶向性，能与肺血管中膜平滑肌细胞特异性结合。（3）葡萄糖基修饰的脂质体包被西地那非长循环时间延长，提高了西地那非的生物利用度，高效液相色谱分析表明，肺组织中西地那非的浓度相对于心脏、脾脏等组织显著升高。（4）葡萄糖基修饰的脂质体能使得肺高压大鼠的平均肺动脉压力降低32%，平滑肌中膜增厚下降41%，抑制右心室心肌细胞肥厚，提高大鼠的生存率。葡萄糖基修饰脂质体的治疗作用可能与靶向运输西地那非上调cGMP，抑制平滑肌细胞增殖，促进线粒体相关凋亡相关。