Li yong nei yuan xing xue guan sheng cheng yi zhi ji vastatin zhi liao jiao zhi mu xi bao liu de yan jiu

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumour in adults. The employment of current standard of care management strategy, that is combining maximum but safe surgical resection, and concomitant chemoradiotherapy, only achieves very modest survival benefits. Antiangiogenesis is a widely studied therapeutic strategy, which restricts the tumour growth by cutting off blood supplement. Although several antiangiogenic agents are now under clinicaland preclinical trials, bevacizumab is still the only one that has been proven to be effective in the treatment of recurrent GBM. However, the clinical use of bevacizumab has encountered the emergence of drug resistance. Its therapeutic benefit is considered limited because of its single pathway targeting. Many researchers believe that the use of broad spectrum angiogenesis inhibitors may leadto better clinical outcomes by overcoming the shortcomings of bevacizumab.Vastatin, the globular non-collagenous 1 (NC1) domain of collagen VIIIα1, was initially proved to inhibit the proliferation and migration of bovine aortic endothelial cells. Although vastatin is similar in origin to other collagen-derived antiangiogenic factors (CDAFs), its antiangiogenic capability in treatment of cancers has not been studied systematically. Our team members previously found that vastatin wasa safe and effective antiangiogenic therapeutic and a potential biomarker for liver cancer. In this thesis, I tried to explore the therapeutic potential of vastatin in treatment of GBM.Using a recombinant adeno-associated virus mediated gene therapy, the antiangiogenic potential of vastatin was first confirmed in vitro that it inhibited proliferation, migration and tube formation of murine microvascular endothelial cells (MECs). These effects were further confirmed using another gene vector (H1) which was subsequently employed for the in vivo studies. H1 is a nanopolymer gene vector has high affinity with the folate receptors on tumour cells. Transfection ofH1/vastatin reduced MEC proliferation in a U87/MEC co-culture system, suggesting a paracrine inhibition. Mechanism studies showed that vastatin caused a wide range of changes in the global gene transcription level in MECs, indicating a broad spectrum of action.Following the establishment of an orthotopic murine GBM model, the H1/DNA polyplexes were injected directly to the tumour area. Treatment induced a significant increase in intracranial mRNA level of the therapeutic gene. Both vastatin and endostatin, a positive control, prolonged the survivals of GBM bearing mice. Immunostaning showed that vastatin decreased microvessel density in the outer layer of the tumour, while decreased cell density and caused abnormal vessel structures inthe centre. No synergistic effect was observed when GBM was treated with the combination of H1/vastatin and temozolomide (TMZ) in this model.Finally, the therapeutic effect of vastatin on a TMZ resistant model was studied. GBM cells with acquired TMZ resistance (ATR) were established by chronic exposure of U87 cells to TMZ. Animals grafted with the U87-ATR cells were proved to be tolerant of TMZ treatment. H1/vastatin injection significantly prolonged the survival in this model. More interestingly, H1/vastatin also resensitized these animals to TMZ treatment. Stem cell related drug resistance was supposed to be disturbed in this process.In conclusion, the present study has demonstrated for the first time that vastatin, a broad spectrum endogenous angiogenesis inhibitor, is of therapeutic benefit in a murine orthotopic GBM model. Vastatin’s capability to reverse TMZ resistance highlights an important area for further research.胶质母细胞瘤(GBM)是成人最常见的恶性原发性脑肿瘤。目前的治疗手段包括了手术切除和放化疗，但是效果仍不能让人满意。与传统的化疗药不同，抗血管生成药物能通过抑制肿瘤内新血管的形成，切断血流供给，达到限制肿瘤生长的目标。贝伐单抗（Bevacizumab)是目前唯一获得批准用于临床GBM治疗的抗血管生成药物。然而Bevacizumab在临床应用中必须面对耐药性产生的问题， 而且因为Bevacizumab只单一性地阻断血管内皮生长因子相关的通路，所以它的治疗效果也受到了一定程度的限制，让肿瘤可以选择替代性的通路来获得新生血管。因此一些研究人员认为，改用多靶点或者广谱的抗血管生成药物，治疗效果应该会更好。Vastatin是VIII型胶原蛋白α1链上的球状非胶原裂解片断。人体内这一类的片段多被证明了具有抗血管生成的功能，它们统称为“源自胶原蛋白的抗血管生成因子”。Vastatin具有抑制牛主动脉内皮细胞增殖和迁移的作用，然而它在抗肿瘤血管生成方面的作用却没有被系统地研究过。我们之前的实验曾经发现Vastatin对肝癌模型中的血管生成具有明显的抑制效果，而本论文将对Vastatin是否同样具有治疗GBM的作用展开研究。在体外，我们首先证明了重组腺相关病毒（rAAV）介导的Vastatin基因治疗能有效抑制MEC的增殖和迁移，并阻止其形成管状结构。我们同时也测试了另一种基因载体H1，以方便后续动物实验的开展。H1是一种纳米聚合物，对肿瘤细胞表面高表达的叶酸受体有高亲和力。H1 介导的Vastatin 基因治疗对肿瘤细胞和MEC都没有直接的作用，但在两种细胞的共培养体系中，Vastatin可以通过旁分泌的方式来抑制MEC的增殖。对机制的研究发现，Vastatin使MEC内基因转录的水平发生了大范围多通路的改变，说明了它的作用具有一定的广谱性。实验进一步研究了Vastatin在小鼠原位GBM 模型中的作用。将H1/DNA 复合物直接注入瘤区可以明显提高颅内相应基因的转录水平。Vastatin和作为阳性对照的Endostatin都能有效地延长GBM小鼠的生存期。免疫组织化学的结果显示Vastatin 能降低肿瘤内部的微血管密度，并诱导组织坏死。这与之前报道过的Endostatin的作用相似。在同一模型上，我们还测试了Vastatin和Temozolomide(TMZ)结合给药的效果，但并没有了现明显的协同作用。实验最后研究了Vastatin在TMZ耐药模型中的治疗效果。通过将U87细胞长期浸泡中含有TMZ的培养基中，我们成功地筛选出了具有TMZ耐药性的GBM细胞。用这些细胞建立的小鼠GBM模型对TMZ的作用不敏感。实验表明，H1/Vastatin基因疗法不仅能够明显延长模型小鼠的生存期，还可以逆转耐药性，使TMZ重新发挥作用。我们推测干细胞相关的耐药性的产生和维持可能在这个过程中受到了影响。上述研究第一次阐明了Vastatin对GBM的治疗效果。Vastatin具有广谱的抗血管特性，能够通过作用于MEC抑制肿瘤内部新血管的生成。Vastatin不仅本身具有治疗作用，还能逆转动物模型对化疗药物的耐受性，因些具有很高的研究价值。相信对Vastatin更一步的探索不但可以拓宽我们对抗血管生成药物的理解，也可能意味着一个新的研究领域的出现。Li, Yi.Thesis (Ph.D.)--Chinese University of Hong Kong, 2014.Includes bibliographical references (leaves 102-110).Abstracts also in Chinese.Title from PDF title page (viewed on 05, January, 2017).Li, Yi.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only.Detailed summary in vernacular field only