本文選題：動(dòng)脈粥樣硬化 + GM1��； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：隨著社會(huì)的進(jìn)步,生活節(jié)奏的加快,人們對(duì)身體健康的忽視,導(dǎo)致不良生活方式的產(chǎn)生和體育鍛煉時(shí)間的減少,以及高脂高糖高蛋白的攝取,致使“三高”癥狀、甚至心血管疾病(如動(dòng)脈粥樣硬化等)的頻發(fā)和早發(fā)。對(duì)于健康生活意識(shí)的傳播和相關(guān)藥物的研發(fā),已經(jīng)得到越來越多的科研團(tuán)隊(duì)的重視。在他汀類降血脂藥物中,洛伐他汀(Lovastatin,LT)是比較普遍的緩解心血管疾病的藥物,但由于其脂溶性的特性,在血液中的溶藥率低,被人體吸收量少;沒有靶向性,不能特異性針對(duì)血管受損區(qū),增加了肝臟的負(fù)擔(dān);沒有藥物緩釋的特性,不能持續(xù)給藥,在血液當(dāng)中停留時(shí)間短等問題。為此,科學(xué)家們已提出了多種策略來解決其所存在的缺陷,利用脂蛋白來包裹他汀類藥物即是比較有潛力的一種策略。脂蛋白是存在于人體血液中的一類內(nèi)源性的脂質(zhì)-蛋白質(zhì)復(fù)合物,具有磷脂單分子層,構(gòu)成了疏水性的核心和親水性的外殼;外表鑲嵌的載脂蛋白具有識(shí)別作用,在血管內(nèi)能靶向性的運(yùn)輸膽固醇。其中高密度脂蛋白(high-density lipoprotein,HDL)具有載脂蛋白A-1(ApoA-1)能被清道夫受體識(shí)別,將周圍組織中的膽固醇運(yùn)送到肝臟代謝并排出到體外,從而降低血脂,是一種抗動(dòng)脈粥樣硬化的脂蛋白,俗稱“血管清道夫”。利用這一特性,人工構(gòu)建重組高密度脂蛋白(rHDL),加以其他修飾,可以達(dá)到靶向性、緩釋性和同源性等目的。以rHDL為藥物載體運(yùn)送他汀類藥物是近幾年興起的研究熱點(diǎn),但在血液滯留時(shí)間和緩釋性等方面還有待提高。利用單唾液酸神經(jīng)節(jié)苷脂(GM1)修飾rHDL有望實(shí)現(xiàn)這個(gè)目標(biāo)。神經(jīng)節(jié)苷脂GM1是內(nèi)源性的細(xì)胞膜組成成分之一。已有研究報(bào)道,脂質(zhì)體表面修飾GM1能延長其在血液循環(huán)中的停留時(shí)間,也能減少肝臟對(duì)脂質(zhì)體的攝取。本課題首次使用GM1修飾的rHDL作為藥物載體來運(yùn)送洛伐他汀,對(duì)該藥物-載體復(fù)合物(GM1-LT-r HDL)進(jìn)行了制備和鑒定以及初步的抗動(dòng)脈粥樣硬化的藥效檢測。(1)利用薄膜分散法制取脂質(zhì)運(yùn)載體,進(jìn)一步用GM1和ApoA-1修飾,并添加藥物后得到GM1-LT-rHDL。外源添加的ApoA-1可以給予重組脂蛋白一定的靶向性,將藥物運(yùn)送到受損斑塊區(qū)。外源添加的GM1能夠插入到脂質(zhì)層,形成類細(xì)胞膜表面結(jié)構(gòu),起到緩慢釋放藥物等的作用,同時(shí)減少肝臟的攝取。(2)通過Zeta粒徑儀和透射電子顯微鏡對(duì)GM1-LT-rHDL的形貌和尺寸進(jìn)行了檢測,測得其粒徑117.1±3.6 nm,Zeta電位41.97±1.57 mV。(3)以硅珠和巨噬細(xì)胞為模型,利用分子間的特異性結(jié)合(GM1/CTB,ApoA-1/anti-ApoA-1),通過激光共聚焦顯微鏡、流式細(xì)胞儀和熒光顯微鏡等儀器確證了GM1-LT-rHDL中含有GM1和ApoA-1分子,進(jìn)一步驗(yàn)證了GM1-LT-rHDL的成功構(gòu)建。(4)通過高效液相色譜儀,測得其包封率為76.55±0.41%,載藥量為3.83±0.02%。(5)體外藥物釋放的實(shí)驗(yàn),驗(yàn)證了GM1-LT-rHDL具有緩慢釋放藥物的能力。(6)體外細(xì)胞實(shí)驗(yàn)證實(shí),GM1-LT-rHDL能夠通過ApoA-1被巨噬細(xì)胞表面的清道夫受體SR-BI識(shí)別,且對(duì)巨噬細(xì)胞吞噬Ox-LDL轉(zhuǎn)變成泡沫細(xì)胞的過程(動(dòng)脈粥樣硬化發(fā)生和發(fā)展的關(guān)鍵步驟)有抑制作用。(7)藥物注射動(dòng)脈粥樣硬化模式小鼠(高脂喂食的ApoE-/-小鼠),通過全自動(dòng)生化儀檢測血脂,以及對(duì)動(dòng)脈用油紅O染色的橫向切片和縱向解剖觀察斑塊區(qū)大小,證實(shí)GM1-LT-rHDL不僅能夠降低小鼠的血脂,而且還能抑制動(dòng)脈斑塊的形成。本課題成功實(shí)現(xiàn)了該藥物-載體復(fù)合物(GM1-LT-rHDL)的制備和鑒定以及初步的抗動(dòng)脈粥樣硬化的藥效檢測,但對(duì)于其在動(dòng)物體內(nèi)的代謝、組織中的分布等有待后續(xù)深入研究。基于GM1修飾的rHDL藥物載體系統(tǒng)不但可運(yùn)送洛伐他汀等治療心血管疾病(如動(dòng)脈粥硬化)的他汀類藥物,還可作為運(yùn)送治療其他疾病的脂溶性藥物的載體系統(tǒng),后續(xù)可在這方面開展進(jìn)一步研究。
[Abstract]:With the progress of the society, the quickening of the rhythm of life, the neglect of the health of the people, the production of bad lifestyle and the decrease of physical exercise time, and the intake of high fat and high protein, and the frequent occurrence and early onset of the "three high" symptoms, even the cardiovascular disease (such as atherosclerosis, etc.). The research and development of sowing and related drugs has been paid more and more attention. In statins, Lovastatin (LT) is the most common drug to alleviate cardiovascular disease. But because of its fat solubility, the drug rate in the blood is low and the body is absorbed less in the human body; there is no target and no specific needle. In the area of vascular damage, the burden of the liver is increased; no drug release characteristics, no continuous delivery and short stay in the blood, scientists have proposed a variety of strategies to solve the defects it exists. The use of lipoprotein to encapsulate statins is a more potential strategy. Lipoprotein is the existence of a strategy. A class of endogenous lipid protein complexes in the human blood, having a phosphatidylcholine single molecular layer that forms a hydrophobic core and a hydrophilic shell; the inlaid apoprotein has a recognition role in targeting the transport of cholesterol in the blood vessels. High-density lipoprotein (HDL) has a fat loaded egg. White A-1 (ApoA-1) can be identified by the scavenger receptor, transporting cholesterol in the surrounding tissue to the liver and expelled to the body, thereby reducing blood lipids. It is a kind of anti atherosclerotic lipoprotein, known as the "blood vessel scavenger,". Using this characteristic, the recombinant high density lipoprotein (rHDL) is artificially constructed and other modifications can be used to target the target. It is a hot spot in recent years to transport statins with rHDL as a drug carrier, but it still remains to be improved in the period of blood retention and sustained release. The use of monaloside Ganglioside (GM1) to modify rHDL is expected to achieve this target. Ganglioside GM1 is an endogenous cell membrane composition. One of the ingredients. It has been reported that the surface modification of liposome GM1 can prolong its retention time in the blood circulation and reduce the liver's uptake of liposomes. For the first time, the GM1 modified rHDL was used as a drug carrier to transport lovastatin, and the drug carrier complex (GM1-LT-r HDL) was prepared and identified as well as preliminary. The anti atherosclerotic efficacy detection. (1) using the thin film dispersion method to take the lipid carrier, further modified by GM1 and ApoA-1, and adding the GM1-LT-rHDL. exogenous ApoA-1 can give the recombinant lipoprotein a certain target and transport the drug to the damaged patch area. The exogenous GM1 can be inserted into the lipid layer. Form the surface structure of the cell membrane, play the role of slow release of drugs and reduce the uptake of liver. (2) the morphology and size of GM1-LT-rHDL were detected by Zeta particle size meter and transmission electron microscope, and the size of the particle was 117.1 + 3.6 nm, the Zeta potential was 41.97 + 1.57 mV. (3), and the silicon beads and macrophages were used as the model. GM1/CTB (ApoA-1/anti-ApoA-1), the GM1 and ApoA-1 molecules in GM1-LT-rHDL were confirmed by laser confocal microscopy, flow cytometry and fluorescence microscopy, and the successful construction of GM1-LT-rHDL was further verified. (4) the encapsulation efficiency was 76.55 + 0.41% and the drug loading was 3.83 + 0.02%. by high performance liquid chromatography. (5) the experiment of drug release in vitro demonstrated that GM1-LT-rHDL has the ability to release drugs slowly. (6) in vitro cell experiments confirmed that GM1-LT-rHDL can be identified by ApoA-1 by the scavenger receptor SR-BI on the surface of macrophages and the process of macrophage phagocytosis of Ox-LDL into a foam cell (a key step in the occurrence and development of atherosclerosis. (sudden) inhibition. (7) the drug injection of atherosclerosis model mice (high fat fed ApoE-/- mice), using a full automatic biochemical analyzer to detect blood lipids, and the transverse section and longitudinal anatomy of the artery oil red O staining to observe the size of the plaque area, GM1-LT-rHDL can not only reduce the blood lipid in mice, but also inhibit atherosclerotic plaque. The preparation and identification of the drug carrier complex (GM1-LT-rHDL) and the preliminary detection of anti atherosclerotic efficacy have been successfully realized. However, the metabolism in animals and the distribution of tissue in the animals need to be further studied. The GM1 modified rHDL drug carrier system can not only transport lovastatin and so on. Statins for the treatment of cardiovascular diseases, such as atherosclerosis, can also be used as a carrier system for the transport of fat soluble drugs for other diseases, and further research in this respect can be carried out in this respect.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R943

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