脂酰化抗菌肽Camel在肿瘤治疗中的应用研究

发布时间：2018-06-06 20:00

本文选题：抗菌肽 + 穿膜肽　；参考：《兰州大学》2017年硕士论文

【摘要】：恶性肿瘤已经成为继心血管疾病之后的第二大致死性疾病,严重危害人类健康、制约社会经济发展。针对恶性肿瘤的治疗药物以细胞毒性药物为主,但是选择性差、伴随较大的毒副作用以及容易出现耐药性等限制了它们的应用。近年来基因治疗也已经成为某些肿瘤的有效治疗方法之一。然而由于核酸分子不能透过细胞膜,所以发展高效安全的核酸药物载体是推进肿瘤基因治疗的关键。抗菌肽(AMPs)因其独特的作用方式而不易出现耐药性,研究发现有些AMPs也具有抗肿瘤活性。AMPs一般是通过破坏肿瘤细胞的细胞膜来杀死肿瘤细胞,也有些AMPs是穿过细胞膜进入细胞后通过作用于细胞器来杀死肿瘤细胞,因此有些AMPs可以有效地穿过细胞膜而作为穿膜肽(CPPs)来应用。十八烷酸修饰可以显著提高AMPs的转染效率,基于此我们实验小组发展了18-melittin和18-K6L9等CPPs,它们具有接近于Lipofectamin 2000的转染活性。Camel是由melittin和cecropin A组成的嵌合AMPs,具有比melittin更强的膜扰动性。同时研究发现Camel也具有穿过细胞膜破坏线粒体导致细胞死亡的抗肿瘤活性。因此,我们研究了Camel的穿膜活性,并且在Camel氨基端接上不同长度(C4、C8、C12、C16、C18)的脂肪酸链来探究脂肪酸链长度对Camel抗肿瘤活性和细胞穿膜活性的影响。我们的研究结果表明Camel以及脂酰化Camel具有很强的抗肿瘤活性。其中接入短脂肪酸链的4-Camel、8-Camel具有快速破坏肿瘤细胞膜的活性,所以具有快速抗肿瘤活性;而接入较长脂肪酸链的16-Camel和18-Camel则失去了快速破坏细胞膜的活性,但是在作用长时间72h后具有更强的抗肿瘤活性,这是可能因为它们自组装进入细胞后通过作用于细胞器而发挥的抗肿瘤作用。对脂酰化Camel穿膜活性的研究结果表明接十二个脂肪酸链后穿膜活性开始增强,16-Camel和18-Camel的穿膜活性最强,其中18-Camel具有比18-melittin更强的转染活性。我们的结果也表明18-Camel是以小窝蛋白介导的内吞为主、以网格蛋白介导的内吞为辅的内吞方式进入细胞的,并且18-Camel可以有效地促进内吞体逃逸达到更强的转染活性。此外,血清对18-Camel的穿膜活性基本没有影响,说明18-Camel具有体内应用的潜力。进一步的研究表明18-Camel可以携带p53质粒进入肿瘤细胞中通过增加p53蛋白的表达来提高抗肿瘤活性,同时18-Camel和p53质粒以及MDM2抑制剂Nultin-3a的协同抗肿瘤效果更强;此外,18-Camel也可以携带survivin si RNA进入MCF-7和MDA-MB-231细胞提高抗肿瘤活性。总之,我们发展了一种高效抗肿瘤多肽(ACPs),它不仅可以通过直接杀死作用抑制肿瘤生长,也可以通过携带基因起到肿瘤基因治疗的作用,为抗肿瘤药物的研发提供了新的策略。
[Abstract]:Malignant tumor has become the second most fatal disease after cardiovascular disease, which seriously endangers human health and restricts social and economic development. Cytotoxic drugs are the main therapeutic drugs for malignant tumors, but their application is limited by poor selectivity, large side effects and drug resistance. In recent years, gene therapy has become one of the effective treatments for some tumors. However, because nucleic acid molecules can not penetrate the cell membrane, the development of efficient and safe nucleic acid drug carriers is the key to promote tumor gene therapy. Antimicrobial peptides (AMPs) are not susceptible to drug resistance due to their unique way of action. Studies have found that some AMPs also have anti-tumor activity. AMPs generally kill tumor cells by destroying the cell membrane of tumor cells. Some AMPs kill tumor cells by acting on organelles after entering the cells through the cell membrane, so some AMPs can effectively cross the cell membrane and be used as transmembrane peptides (CPPs). Octadecanoic acid modification can significantly improve the transfection efficiency of 18-melittin and 18-K6L9. CPPs, such as 18-melittin and 18-K6L9, have the same transfection activity as Lipofectamin 2000. Camel is a chimeric AMPs composed of melittin and cecropin A, and has stronger membrane disturbance than melittin. It was also found that Camel also had the anti-tumor activity of destroying mitochondria through the cell membrane and resulting in cell death. Therefore, we studied the transmembrane activity of Camel, and attached the fatty acid chains of different lengths to Camel amino terminal to explore the effect of the length of fatty acid chain on Camel's antitumor activity and cell membrane penetration activity. Our results show that Camel and acylated Camel have strong anti-tumor activity. 4-Camelan8-Camel with short fatty acid chains had rapid anti-tumor activity due to its rapid destruction of tumor cell membrane, while 16-Camel and 18-Camel with longer fatty acid chains lost the activity of rapid destruction of cell membrane. However, the antitumor activity was stronger after 72 h of treatment, which may be due to their antitumor effect by acting on organelles after self-assembly into the cells. The results showed that the transmembrane activity of acylated Camel began to enhance the transmembrane activity of 16-Camel and 18-Camel, and 18-Camel had stronger transfection activity than 18-melittin. Our results also showed that 18-Camel was mainly endocytosis mediated by fossa protein and endocytosis supplemented by grid protein. 18-Camel could effectively promote endocytosis escape to achieve stronger transfection activity. In addition, serum had no effect on the transmembrane activity of 18-Camel, indicating that 18-Camel has the potential to be used in vivo. Further studies showed that 18-Camel could carry p53 plasmid into tumor cells to increase the anti-tumor activity by increasing the expression of p53 protein, and the synergistic effect of 18-Camel and p53 plasmids and MDM2 inhibitor Nultin-3a was stronger. In addition, C18-Camel could also carry survivin si RNA into MCF-7 and MDA-MB-231 cells to enhance antitumor activity. In a word, we have developed an effective antitumor polypeptide, ACPsN, which can not only inhibit tumor growth through direct killing, but also provide a new strategy for the development of antitumor drugs by carrying genes to play the role of tumor gene therapy.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.5

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