本文選題：肝癌靶向 + 納米粒子　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的肝細(xì)胞肝癌(hepatocellular carcinoma,HCC,簡(jiǎn)稱肝癌)是占據(jù)全球癌癥死因第三位的常見惡性腫瘤。大多數(shù)病人確診時(shí)已處晚期,失去手術(shù)機(jī)會(huì)。傳統(tǒng)的化療方法效果差且毒副作用較大,因此,提高藥物的治療作用同時(shí)減少其副反應(yīng)成為研究的重點(diǎn)。肝癌的發(fā)生、發(fā)展和轉(zhuǎn)移是一個(gè)多基因共同參與的過程,將一些特定的基因如p DNA、si RNA或micro RNA導(dǎo)入肝癌細(xì)胞繼而在基因?qū)用娓蓴_腫瘤的發(fā)生發(fā)展已成為肝癌治療的新思路。將化療藥物與基因療法相結(jié)合,利用兩者的不同機(jī)制可以發(fā)揮協(xié)同作用。然而,這種聯(lián)合療法最大的挑戰(zhàn)在于如何實(shí)現(xiàn)藥物和基因的共載并將二者靶向運(yùn)送至肝癌組織細(xì)胞。多功能納米載體可以作為藥物/基因的共載介質(zhì)。本研究以阿霉素(doxorubicin,DOX)和寡聚核苷酸(oligo RNA)分別作為藥物和基因模型,構(gòu)建了一種同時(shí)攜載DOX和oligo RNA的超分子納米體系,并探討了其在體內(nèi)外實(shí)驗(yàn)中的肝癌靶向性,為肝癌的臨床治療提供新的方向和方法。方法通過希夫堿反應(yīng)制備出β-環(huán)糊精(β-cyclodextrin,β-CD)和多聚-L-賴氨酸(poly-L-lysine,PLL)結(jié)合的天然生物材料β-環(huán)糊精接枝聚賴氨酸(命名為PLCD),采用傅立葉紅外分析及核磁共振氫譜表征了PLCD的化學(xué)結(jié)構(gòu);應(yīng)用芘的熒光實(shí)驗(yàn)及瓊脂糖凝膠電泳阻滯實(shí)驗(yàn)分別考察了不同β-CD取代度的PLCD攜載疏水性分子及復(fù)合oligo RNA的能力;采用透析法制備PLCD/DOX復(fù)合物,將其與oligo RNA混合,形成PLCD/DOX/oligo RNA復(fù)合物(簡(jiǎn)稱PDR);采用共孵育的方法將透明質(zhì)酸(hyaluronic acid,HA)引入到PDR納米粒子表面,形成具有肝癌靶向性的超分子納米粒子(簡(jiǎn)稱為HPDR)。應(yīng)用動(dòng)態(tài)透析法測(cè)定PDR和HPDR兩種納米體系中的DOX在不同p H值環(huán)境下的體外釋放;應(yīng)用流式細(xì)胞術(shù)(flow cytometry)和激光共聚焦掃描顯微鏡(confocal laser scanning microscope,CLSM)檢測(cè)CD44分子在肝癌細(xì)胞系MHCC-97H和Hep G2表面的表達(dá)情況及HPDR納米粒子靶向遞送DOX和熒光標(biāo)記的oligo RNA(FAM-RNA)至肝癌細(xì)胞的能力;應(yīng)用CCK-8(Cell Counting Kit-8)試劑檢測(cè)PLCD和HA/PLCD的生物安全性,并比較PDR和HPDR納米粒子的細(xì)胞毒性作用。構(gòu)建MHCC-97H皮下荷瘤裸鼠模型,經(jīng)尾靜脈注射給藥,采用小動(dòng)物活體成像系統(tǒng)檢測(cè)HPDR在小鼠體內(nèi)的分布。結(jié)果1.成功制備了三種不同β-CD取代度的PLCD,取代度分別為7.3%、12.9%和25.1%。2.PLCD可以有效包載DOX,其載藥量為11.0%,包封率為41.2%。3.當(dāng)N/P為30/1時(shí),PLCD/DOX與oligo RNA復(fù)合形成穩(wěn)定的納米粒子,其粒徑為168.9 nm,表面電荷為+38.7 m V。4.當(dāng)HA/PDR的質(zhì)量比為3/6時(shí),HA可成功包裹于PDR納米粒子表面形成負(fù)電性的殼層,形成的HPDR納米粒子成規(guī)則球形,具有典型的核-殼結(jié)構(gòu),粒徑為195.8 nm,表面電荷為-22.7 m V。5.肝癌細(xì)胞系MHCC-97H和Hep G2表面均高表達(dá)CD44分子。6.HPDR納米粒子可通過HA-CD44介導(dǎo)的內(nèi)吞作用將DOX和RNA靶向遞送至肝癌細(xì)胞,并表現(xiàn)出較強(qiáng)的細(xì)胞毒性。7.體內(nèi)實(shí)驗(yàn)表明,與PDR納米粒子相比,HPDR在腫瘤部位蓄積更多而在肝臟組織中蓄積較少,說明HPDR納米粒子在體內(nèi)具有良好的肝癌靶向性。結(jié)論本研究中制備的HPDR超分子納米粒子結(jié)構(gòu)穩(wěn)定,在體內(nèi)外均表現(xiàn)出良好的肝癌靶向性,有望在藥物與基因聯(lián)合治療肝癌方面發(fā)揮巨大的潛力。
[Abstract]:Objective: hepatocellular carcinoma (hepatocellular, carcinoma, HCC, HCC) is a common malignant tumor in the world occupy the third cause of cancer death. Most patients are diagnosed at advanced stage and lost the chance of operation. The effect of the traditional chemotherapy and toxic side effects, therefore, provided high therapeutic effects of drugs and reduce the side-effects become. The key. The occurrence of liver cancer, development and transfer process is a multi gene involved, some specific genes such as P DNA, Si RNA or micro RNA into liver cancer cell and gene level interference on the occurrence and development of tumor has become a new way of thinking in the treatment of liver cancer. The chemotherapy drug and gene therapy combination the use of different machine. We can play a synergistic role. However, the biggest challenge is how to realize the combination therapy of drugs and genes were loaded and the two targeted delivery to liver cancer cells. Multifunctional nanocarriers can be used as drug / gene carrying medium. In this study, adriamycin (doxorubicin, DOX and oligonucleotide (oligo RNA) as) respectively. Drug and gene model, constructed a simultaneously carrying DOX and oligo RNA nano supramolecular system, and discusses the in vivo and in the liver targeting, provide new directions and methods for clinical treatment of hepatocellular carcinoma. Methods by Schiff base reaction for the preparation of beta cyclodextrin (3 -cyclodextrin. Beta -CD) and poly -L- lysine (poly-L-lys Ine, PLL) with the natural biological materials of beta cyclodextrin grafted poly lysine (named PLCD), were used to characterize the chemical structure of PLCD by Fu Liye infrared analysis and NMR; fluorescence experiments and agarose gel electrophoresis retardation test application of pyrene were investigated with different degree of substitution of beta -CD PLCD carrying hydrophobic molecular and composite oligo RNA The preparation of PLCD/DOX composite material; by the dialysis method, the oligo and RNA mixed formation of PLCD/DOX/oligo RNA complex (PDR); using the method of CO incubation of hyaluronic acid (hyaluronic acid HA) is introduced to the surface of PDR nanoparticles, forming a supramolecular of liver targeted nanoparticles (HPDR) the application of dynamic dialysis method. Determination of PDR and HPDR in the system of two kinds of nano DOX in different P H release environment in vitro; application of flow cytometry (flow cytometry) and laser scanning confocal microscope (confocal laser scanning microscope, CLSM) detection of CD44 molecules to deliver DO expression in target MHCC-97H and Hep and HPDR nanoparticles on the surface of G2 hepatocellular carcinoma cell lines Oligo RNA X (FAM-RNA) and fluorescence labeling ability to hepatocellular carcinoma cells; application of CCK-8 (Cell Counting Kit-8) biological safety detection reagent PLCD and HA/PLCD, cytotoxicity and comparison of PDR and HPDR nanoparticles. Construct MHCC-97H subcutaneous xenograft models in nude mice by tail vein injection, administered by small animal in vivo the imaging detection system HPDR Tissue distribution in mice. Results 1. were successfully prepared three different beta -CD substituted PLCD, the degree of substitution were 7.3%, 12.9% and 25.1%.2.PLCD can be effectively encapsulated DOX, the drug loading was 11%, the encapsulation rate of 41.2%.3. when N/P is 30/1, PLCD/DOX and oligo RNA to form stable composite nanoparticles. The diameter is 168.9 NM, the surface charge is +38. 7 m V.4. when the mass ratio of HA/PDR to 3/6, HA can be successfully encapsulated in PDR nanoparticles formed on the surface of the negatively charged shell, the formation of HPDR nanoparticles into regular spherical core-shell structure typical of the particle size of 195.8 nm -22.7 m V.5., the surface charge of hepatocellular carcinoma cell line MHCC-97H and Hep G2 were high surface the expression of CD44.6.HPDR nanoparticles Endocytosis can be mediated by HA-CD44 DOX and RNA targeted delivery to liver cancer cells, and showed that the cytotoxicity of.7. in vivo is stronger, compared with PDR nanoparticles, HPDR at the tumor site and accumulated more in liver tissue accumulation less, indicating that HPDR nanoparticles in vivo hepatocellular carcinoma has good target. The conclusions of this study HPDR nanoparticles were prepared by supramolecular structure stability, in vitro and in vivo showed good liver targeting, is expected to play a huge potential in drug and gene therapy of liver cancer.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 彭穗;王晶;彭振維;許麗霞;郭宇;匡銘;;攜帶缺氧誘導(dǎo)因子-1α-siRNA的葉酸靶向化的磁性納米復(fù)合物抑制肝癌干細(xì)胞增殖的實(shí)驗(yàn)研究[J];中華細(xì)胞與干細(xì)胞雜志(電子版);2016年01期

2 William C.S.Meng;Yunlong Pan;Xiaoxu Zhao;;Epirubicin-gold nanoparticles suppress hepatocellular carcinoma xenograft growth in nude mice[J];The Journal of Biomedical Research;2015年06期

3 Jaleh Varshosaz;Maryam Farzan;;Nanoparticles for targeted delivery of therapeutics and small interfering RNAs in hepatocellular carcinoma[J];World Journal of Gastroenterology;2015年42期

4 唐勇;李堅(jiān);張陽德;;具有肝靶向功能的半乳糖化紫杉醇長(zhǎng)循環(huán)納米脂質(zhì)體抑瘤作用研究[J];中國(guó)現(xiàn)代醫(yī)學(xué)雜志;2012年09期

5 陳建;;納米制劑在抗肝癌藥物中的應(yīng)用[J];中國(guó)醫(yī)藥指南;2012年03期

6 盛世厚;于惠秋;劉銅軍;柳時(shí);鄭勇輝;胡秀麗;宋祥福;;鍵合紫杉醇納米膠束對(duì)H22肝癌小鼠的體內(nèi)療效評(píng)估[J];應(yīng)用化學(xué);2011年11期

7 丁茹茹;張英鴿;;納米粒子在肝癌靶向治療中的應(yīng)用研究進(jìn)展[J];生物技術(shù)通訊;2008年02期

8 彭健;劉鑫;王榮兵;劉路;鄔力祥;;5-氟尿嘧啶磁性納米脂質(zhì)體在大鼠肝癌模型體內(nèi)的靶向分布[J];中國(guó)現(xiàn)代醫(yī)學(xué)雜志;2007年24期

9 賀連香;張陽德;;納米粒在肝癌靶向治療中的研究進(jìn)展[J];中國(guó)醫(yī)學(xué)工程;2007年03期

10 申良方;王欣;王騁;趙凱;劉新菊;申海菊;;阿霉素聚氰基丙烯酸正丁酯納米粒粒徑對(duì)肝靶向性的影響[J];中南大學(xué)學(xué)報(bào)(醫(yī)學(xué)版);2006年05期

，

本文編號(hào)：2046207