穿膜肽RDP引导DNA的靶脑转运及跨膜机制探究

发布时间：2018-05-25 05:04

本文选题：血脑屏障 + 穿膜肽RDP　；参考：《西南大学》2014年硕士论文

【摘要】：血-脑屏障(BBB)的存在是CNS药物发展缓慢的主要原因,它使得很多具有治疗潜力的药物难以发挥作用。因此,脑部疾病治疗的关键在于寻找克服BBB或促进药物透过BBB使之在脑内能达到有效浓度的方法。传统的脑部疾病治疗方法立体定位手术,脑内注射药物,基因载体的直接输送具有高侵入性,因其扩散范围小、药物维持时间短、表达效率低等弊端得不到广泛的应用。现今提出的一种新技术利用病毒或非病毒载体可将大分子活性药物携带入脑。病毒载体有明显的副作用且不易进行靶向改造,反之非病毒载体具有了生物可降解性、生物相容性等优势,在治疗CNS疾病中展现出了良好的应用前景。已有研究报道证实多肽作为非病毒载体可携带寡核苷酸、siRNA或DNA穿过BBB,实现靶脑转运。本实验研究来源于狂犬病毒糖蛋白的一个新型衍生肽(RVG-derived peptide, RDP),是否能够引导DNA进行靶脑转运及跨膜机理的初步探究。在RDP引导DNA靶脑转运的实验中首先将RDP与含有报告基因LacZ和EGFP的质粒以非共价键的方式进行连接,通过琼脂糖凝胶电泳阻滞实验和圆二色谱确定RDP与DNA复合物的最佳比例以及相互作用。体内实验中,将实验小鼠分为实验组(尾静脉注射最佳比例混合的RDP和DNA复合物),对照组(尾静脉注射DNA)和空白对照组(尾静脉注射生理盐水),固定时间后处死小鼠,取其脑、肝、肾等组织,冷冻切片后进行X-gal染色和荧光观察来确定目的蛋白在各组织中的表达情况。实验结果表明,RDP/pDNA组小鼠的脑、肾、脾可检测到目的蛋白,脑中目的蛋白含量最多。对照组的目的蛋白只有出现在肾、脾组织中。因此证明RDP可作为基因治疗的一种合适的靶脑载体,在未来的非病毒载体应用中可能具有较大的潜力。在最后体外跨膜机制实验研究中,我们发现实验的HeLa、CHO口SH-SY5Y细胞中RDP只对神经细胞SH-SY5Y具有特异性。在4度和37度条件下进行穿透实验,RDP和RDP-EGFP在37度可以进入细胞而4度不能,表明RDP进入细胞具有能量依赖性。将内吞抑制剂秋水仙素加入细胞中,发现明显的阻断作用,因此判断RDP和RDP-EGFP进入方式可能为包吞作用而不是直接穿透。接下来我们利用Rho标记的RDP和RDP-EGFP的竞争性抑制作用以及神经递质对RDP入脑的阻断作用证明了包吞作用的介导受体并非烟碱乙酰胆碱受体,而可能是与其结构相似的GABA受体。
[Abstract]:The existence of blood-brain barrier (BBB) is the main reason for the slow development of CNS drugs, which make it difficult for many drugs with therapeutic potential to function. Therefore, the key to the treatment of brain diseases is to find ways to overcome BBB or to promote drugs to achieve effective concentration in the brain through BBB. The traditional treatment methods of brain diseases, such as stereotactic surgery, intracerebral injection of drugs and direct delivery of gene vectors, are not widely used because of their small diffusion range, short duration of drug maintenance, low expression efficiency and so on. A new technique is proposed to carry macromolecular active drugs into the brain using viral or non-viral vectors. The virus vector has obvious side effects and is not easy to target modification. On the contrary, the non-virus vector has the advantages of biodegradability and biocompatibility, which shows a good prospect in the treatment of CNS disease. It has been reported that polypeptides as non-viral vectors can carry oligonucleotide siRNA or DNA across BBBs for target brain transport. This study was conducted to investigate whether RVG-derived peptide (RDPN), a new derivative peptide of rabies virus glycoprotein, can guide the target brain transport and transmembrane mechanism of DNA. In the RDP guided DNA target brain transport experiment, RDP was first connected with plasmids containing the reporter genes LacZ and EGFP in a non-covalent manner. The optimum ratio and interaction of RDP and DNA complex were determined by agarose gel electrophoresis and circular dichroism. In vivo, the experimental mice were divided into experimental group (tail vein injection of the best proportion of RDP and DNA complex), control group (tail vein injection of DNA) and blank control group (tail vein injection of normal saline). After fixed time, the mice were killed and their brains were taken out. The expression of target protein in liver and kidney was determined by X-gal staining and fluorescence observation. The results showed that the target protein could be detected in brain, kidney and spleen of RDP / pDNA group, and the content of target protein was the most in brain. The target protein of the control group was only present in the kidney and spleen tissues. Therefore, RDP may be a suitable target brain vector for gene therapy, and may have great potential in the application of non-viral vectors in the future. In the final study of transmembrane mechanism in vitro, we found that RDP in SH-SY5Y cells was specific to neural SH-SY5Y only. RDP and RDP-EGFP could enter the cells at 37 degrees but not at 4 degrees, indicating that RDP entry into the cells was energy-dependent. The endocytosis inhibitor colchicine was added to the cells and the blocking effect was found. It was concluded that the entry mode of RDP and RDP-EGFP might be the entrapment rather than the direct penetration. Then we use the competitive inhibition of RDP and RDP-EGFP labeled by Rho and the blocking effect of neurotransmitters on RDP into the brain to prove that the mediated receptors of entrapment are not nicotinic acetylcholine receptors but may be GABA receptors similar to its structure.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

【参考文献】