本文選題：肝靶向 + 多功能信封式納米裝置��； 參考：《青島大學(xué)》2015年碩士論文

【摘要】：本課題制備了一種轉(zhuǎn)運(yùn)si RNA的非病毒雙靶向長(zhǎng)循環(huán)多功能信封式納米裝置(Multifunction Envelop-type Nano Device,MEND)。MEND為復(fù)合納米脂質(zhì)體,同時(shí)具有脂質(zhì)體和納米粒的特性,通過(guò)對(duì)其脂質(zhì)成分進(jìn)行修飾,達(dá)到長(zhǎng)循環(huán)和肝癌細(xì)胞雙重靶向的目的,轉(zhuǎn)染效率提高。修飾后的脂質(zhì)分子結(jié)構(gòu)為:甘草次酸(Glycyrrhetinic,GA)-聚乙二醇(Polyethylene glycol,PEG)-肽(Peptide,Pp,基質(zhì)金屬蛋白酶MMP的底物)-二油酰磷脂酰乙醇胺(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine,DOPE)。各成分通過(guò)酰胺反應(yīng)結(jié)合,核磁共振氫譜法和質(zhì)譜法進(jìn)行結(jié)構(gòu)鑒定;PLL壓縮的si RNA溶液水化脂質(zhì)體薄膜后,得到si RNA-MEND。依次利用透射電鏡、動(dòng)態(tài)光散射法和超濾離心法,檢測(cè)si RNA-MEND的形態(tài)、粒徑電位以及包封率;體外酶解實(shí)驗(yàn)考察si RNA-MEND在血漿中的穩(wěn)定性;采用MTT法和熒光標(biāo)記法,考察si RNA-MEND對(duì)肝癌BEL-7402細(xì)胞生長(zhǎng)抑制和轉(zhuǎn)染效率;利用K-Ras-si RNA-MEND對(duì)BEL-7402細(xì)胞轉(zhuǎn)染,考察細(xì)胞侵襲轉(zhuǎn)移和基因沉默。結(jié)果表明修飾物GA-PEG-Pp-DOPE成功合成;si RNA-MEND粒子呈光滑的球形,具有明顯的脂質(zhì)雙分子層和指紋結(jié)構(gòu),其粒徑分布范圍為163.5±20.0nm,平均電位為0.614±0.05m V;MEND對(duì)si RNA的包封率為82.8%。酶解實(shí)驗(yàn)表明MEND可以保護(hù)si RNA免受血漿降解長(zhǎng)達(dá)120h,是裸si RNA的40倍;修飾成分中的肽可以被MMP-2特異性降解。MEND的細(xì)胞毒性小,在MEND轉(zhuǎn)染下細(xì)胞的生長(zhǎng)平均生長(zhǎng)率為87.03±4.65%,si RNA-MEND可以高效率的轉(zhuǎn)染進(jìn)入細(xì)胞質(zhì)。由K-Ras-si RNA-MEND轉(zhuǎn)染的細(xì)胞,其侵襲轉(zhuǎn)移能力明顯下降,K-Ras蛋白的表達(dá)水平比未轉(zhuǎn)染細(xì)胞降低78.61倍。MEND的制備工藝簡(jiǎn)單,對(duì)si RNA具有較強(qiáng)的保護(hù)作用,可以明顯延長(zhǎng)si RNA在血液循環(huán)中的時(shí)間,轉(zhuǎn)染效率較高。
[Abstract]:In this paper, a novel multifunction envelope device (multifunction Envelop-type Nano DeviceMend), which transports siRNA, was prepared as a composite nano-liposome with the properties of liposome and nanoparticles. The efficiency of transfection was improved with the aim of double targeting of long circulation and hepatoma cells. The modified lipids were composed of glycyrrhetinic acid (GA) -polyethylene glycoline (PEG) -peptide (PeptidePp) and dioleoyl-sn-glycero-3-phosphoanolaminedoPE (1-dioleoyl-sn-glycero-3-phosphoanolamine). The structures of the liposome films were identified by hydrogen NMR and mass spectrometry. Si RNA-MEND was obtained after hydration of liposome membrane in the solution of si RNA compressed by PLL. The morphology, particle size potential and encapsulation efficiency of siRNA-MEND were detected by transmission electron microscopy, dynamic light scattering and ultrafiltration centrifugation. The stability of siRNA-MEND in plasma was investigated by enzymolysis in vitro. The growth inhibition and transfection efficiency of BEL-7402 cells were investigated by siRNA-MEND, and the invasion, metastasis and gene silencing of BEL-7402 cells were studied by K-Ras-si RNA-MEND transfection. The results showed that the modified GA-PEG-Pp-DOPE successfully synthesized simium-RNA-MEND particles with smooth globular shape, obvious lipid bimolecular layer and fingerprint structure. The particle size distribution range was 163.5 鹵20.0nm.The average potential was 0.614 鹵0.05m VnMEND and the encapsulation efficiency of siRNA-MEND to si RNA was 82.8 folds. The results of enzymatic hydrolysis showed that met could protect siRNA from degradation of plasma for 120 hours, 40 times as much as bare siRNA, and the peptides in modified components could be degraded by MMP-2 specifically. The average growth rate of the cells transfected with met was 87.03 鹵4.65 and siRNA-MEND could transfect into the cytoplasm efficiently. The invasiveness and metastasis ability of the cells transfected with K-Ras-si RNA-MEND was reduced 78.61 times than that of untransfected cells. The time of si RNA in blood circulation was prolonged, and the transfection efficiency was higher.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R450

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 魏秀莉,吳偉,徐惠南;非病毒基因遞送系統(tǒng)的靶向性研究[J];復(fù)旦學(xué)報(bào)(醫(yī)學(xué)版);2005年03期

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本文編號(hào)：2106596