本文選題：血腦屏障 + 穿膜肽RDP��； 參考：《西南大學(xué)》2014年碩士論文

【摘要】：血-腦屏障(BBB)的存在是CNS藥物發(fā)展緩慢的主要原因,它使得很多具有治療潛力的藥物難以發(fā)揮作用。因此,腦部疾病治療的關(guān)鍵在于尋找克服BBB或促進(jìn)藥物透過(guò)BBB使之在腦內(nèi)能達(dá)到有效濃度的方法。傳統(tǒng)的腦部疾病治療方法立體定位手術(shù),腦內(nèi)注射藥物,基因載體的直接輸送具有高侵入性,因其擴(kuò)散范圍小、藥物維持時(shí)間短、表達(dá)效率低等弊端得不到廣泛的應(yīng)用。 現(xiàn)今提出的一種新技術(shù)利用病毒或非病毒載體可將大分子活性藥物攜帶入腦。病毒載體有明顯的副作用且不易進(jìn)行靶向改造,反之非病毒載體具有了生物可降解性、生物相容性等優(yōu)勢(shì),在治療CNS疾病中展現(xiàn)出了良好的應(yīng)用前景。 已有研究報(bào)道證實(shí)多肽作為非病毒載體可攜帶寡核苷酸、siRNA或DNA穿過(guò)BBB,實(shí)現(xiàn)靶腦轉(zhuǎn)運(yùn)。本實(shí)驗(yàn)研究來(lái)源于狂犬病毒糖蛋白的一個(gè)新型衍生肽(RVG-derived peptide, RDP),是否能夠引導(dǎo)DNA進(jìn)行靶腦轉(zhuǎn)運(yùn)及跨膜機(jī)理的初步探究。 在RDP引導(dǎo)DNA靶腦轉(zhuǎn)運(yùn)的實(shí)驗(yàn)中首先將RDP與含有報(bào)告基因LacZ和EGFP的質(zhì)粒以非共價(jià)鍵的方式進(jìn)行連接,通過(guò)瓊脂糖凝膠電泳阻滯實(shí)驗(yàn)和圓二色譜確定RDP與DNA復(fù)合物的最佳比例以及相互作用。體內(nèi)實(shí)驗(yàn)中,將實(shí)驗(yàn)小鼠分為實(shí)驗(yàn)組(尾靜脈注射最佳比例混合的RDP和DNA復(fù)合物),對(duì)照組(尾靜脈注射DNA)和空白對(duì)照組(尾靜脈注射生理鹽水),固定時(shí)間后處死小鼠,取其腦、肝、腎等組織,冷凍切片后進(jìn)行X-gal染色和熒光觀察來(lái)確定目的蛋白在各組織中的表達(dá)情況。實(shí)驗(yàn)結(jié)果表明,RDP/pDNA組小鼠的腦、腎、脾可檢測(cè)到目的蛋白,腦中目的蛋白含量最多。對(duì)照組的目的蛋白只有出現(xiàn)在腎、脾組織中。因此證明RDP可作為基因治療的一種合適的靶腦載體,在未來(lái)的非病毒載體應(yīng)用中可能具有較大的潛力。 在最后體外跨膜機(jī)制實(shí)驗(yàn)研究中,我們發(fā)現(xiàn)實(shí)驗(yàn)的HeLa、CHO口SH-SY5Y細(xì)胞中RDP只對(duì)神經(jīng)細(xì)胞SH-SY5Y具有特異性。在4度和37度條件下進(jìn)行穿透實(shí)驗(yàn),RDP和RDP-EGFP在37度可以進(jìn)入細(xì)胞而4度不能,表明RDP進(jìn)入細(xì)胞具有能量依賴性。將內(nèi)吞抑制劑秋水仙素加入細(xì)胞中,發(fā)現(xiàn)明顯的阻斷作用,因此判斷RDP和RDP-EGFP進(jìn)入方式可能為包吞作用而不是直接穿透。接下來(lái)我們利用Rho標(biāo)記的RDP和RDP-EGFP的競(jìng)爭(zhēng)性抑制作用以及神經(jīng)遞質(zhì)對(duì)RDP入腦的阻斷作用證明了包吞作用的介導(dǎo)受體并非煙堿乙酰膽堿受體,而可能是與其結(jié)構(gòu)相似的GABA受體。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R965

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