本文關鍵詞： 阿爾茨海默癥 聚乳酸羥基乙酸共聚物 CRT β淀粉樣蛋白　出處：《寧夏大學》2017年碩士論文　論文類型：學位論文

【摘要】：阿爾茨海默癥(Alzheimer'sdisease,AD)作為一種目前致死率極高的疾病已經嚴重威脅到了人們的公共健康問題,受到越來越多的關注。其常見的病理特征包括老年斑、神經元纖維異常纏結和神經元胞外高密度β淀粉樣蛋白(amyloid β,Aβ)的沉積。雖然目前已有多種治療藥物上市,但由于存在藥物可溶性差,生物可溶性低,難以穿越血腦屏障(blood-brain barrier,BBB)等問題,致使AD的治療現狀仍不容樂觀。納米顆粒載體因其在體內體外實驗中均易被檢測到因此用于AD的診斷治療日益受到重視。因此,我們希望設計出一種可以穿越BBB靶向腦部并積極發(fā)揮攜帶藥物功能的新型納米藥物載體。聚乳酸輕基乙酸共聚物(Poly(lactic-co-glycolicacid),PLGA)是一種生物可降解的高分子微球,具有良好的生物相容性,應用前景廣闊,因此我們選擇PLGA為基礎納米顆粒藥物載體,并將姜黃素與環(huán)肽S1包裹進納米顆粒內腔,將制得的納米顆粒命名為NP-S1+Cur。姜黃素是一種可以抑制Aβ毒性的天然化合物,而S1可以通過影響APP的剪切過程有效降低Aβ生成,二者對AD的致病機制均有一定的積極影響。隨后我們選擇用穿膜肽CRT修飾在PLGA納米顆粒表面(CRT-NP-S1+Cur),因為CRT是一種與鐵生物性能相似的多肽,可以靶向性結合轉鐵蛋白與轉鐵蛋白受體復合物,增強納米顆粒穿越BBB的能力。通過透射電鏡(Transmission electron microscope,TEM)觀察納米顆粒形態(tài),動態(tài)光散射法(Dynamic light scattering,DLS)確定耦連CRT前后納米顆粒的大小分別為128.6 nm and 139.8 nm,包封率分別為 23.2 ± 5.3%and 21.4 ± 6.9%,載藥量分別為 0.54 ± 0.04%and 0.42 ±0.06%;利用MTT毒性實驗分析納米顆粒的生物毒性;同時,體外模擬血腦屏障實驗和體內活體成像實驗表明,CRT-NP-Sl+Cur具有更強的穿越BBB的能力。動物行為學和病理學研究表明,經過對AD轉基因小鼠21天腹腔注射PLGA納米顆粒后,能夠改善AD鼠的空間記憶能力,小鼠腦內老年斑數量有所減少,小膠質細胞和星型膠質細胞活化水平、突觸素水平均有改善。用試劑盒對實驗小鼠腦部進行分析發(fā)現,PLGA納米顆粒可以增強腦內超氧化物歧化酶(SOD)的活性,降低Aβ和活性氧(ROS)水平,促炎性細胞因子(TNF-α和IL-6)的表達,與NP-S1+Cur相比,CRT-NP-S1+Cur的作用效果更顯著。以上結果表明,本實驗設計的新型納米藥物載體具有穿越BBB并靶向腦部,從而高效發(fā)揮姜黃素抗氧化、抗炎癥作用及S1抑制Aβ生成、降低Aβ毒性的作用,對AD的治療具有潛在的應用價值。
[Abstract]:Alzheimer's disease (AD), a disease with a high mortality rate, has become a serious threat to public health and has attracted increasing attention. Its common pathological features include senile plaques. Abnormal tangles of neuron fibers and deposition of amyloid 尾 -A 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid 尾 -amyloid in neurons. It is difficult to cross the blood-brain barrier and BBB, so the current status of AD treatment is still not optimistic. The nanoparticles carrier is easy to be detected in vivo and in vitro, so the diagnosis and treatment of AD has been paid more and more attention. We hope to design a novel nano-drug carrier that can traverse the brain of BBB and play an active role in carrying drugs. Poly (lactic acid-co-glycolic acid) is a biodegradable polymer microsphere with good biocompatibility. Therefore, we chose PLGA as the base drug carrier and encapsulated curcumin and cyclic peptide S1 into the intracavity of nanoparticles. The prepared nanoparticles were named NP-S1 Cur.Curcumin is a natural compound that can inhibit the toxicity of A 尾, and S1 can effectively reduce A 尾 formation by affecting the shearing process of APP. Both of them have a positive effect on the pathogenesis of AD. Subsequently, we chose to modify CRT-NP-S1 Curan on the surface of PLGA nanoparticles with transmembrane peptide CRT, because CRT is a polypeptide similar to the biological properties of iron. It can target the complex of transferrin and transferrin receptor to enhance the ability of nanoparticles to cross BBB. The morphology of nanoparticles was observed by transmission electron microscopetem (TEM). The size of nanoparticles before and after coupling CRT was 128.6 nm and 139.8 nm, the entrapment efficiency was 23.2 鹵5.3 and 21.4 鹵6.9, and the drug loading was 0.54 鹵0.04 and 0.42 鹵0.06 respectively. The biotoxicity of nanoparticles was analyzed by MTT toxicity test. At the same time, in vitro and in vivo imaging experiments showed that CRT-NP-Sl Cur had a stronger ability to cross BBB. Animal behavioral and pathological studies showed that after 21 days of intraperitoneal injection of PLGA nanoparticles into AD transgenic mice, CRT-NP-Sl Cur had stronger ability to cross BBB. It could improve the spatial memory ability of AD mice. The number of senile plaques in the brain of AD mice was decreased, and the activation levels of microglia and astrocytes were decreased. The level of synaptophysin was improved. The results showed that PLGA nanoparticles could enhance the activity of superoxide dismutase (SOD), decrease the levels of A 尾 and reactive oxygen species (Ros), and promote the expression of inflammatory cytokines TNF- 偽 and IL-6. Compared with NP-S1 Cur, the effect of CRT-NP-S1 Cur was more significant. The results showed that the novel drug carrier could travel through BBB and target the brain, thus effectively exerting curcumin antioxidation, anti-inflammatory effect and S1 inhibiting A 尾 production. Reducing the toxicity of A 尾 has potential application value in the treatment of AD.
【學位授予單位】：寧夏大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R749.16

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