本文關(guān)鍵詞：VA修飾PEG-PCL納米膠束載喜樹(shù)堿選擇性抑制活化態(tài)肝星狀細(xì)胞糖酵解行為及小鼠肝纖維化的研究　出處：《西南交通大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 缺氧因子(HIF-1-α) 糖酵解 纖維化表型 肝星狀細(xì)胞 喜樹(shù)堿 肝纖維化

【摘要】：活化態(tài)肝星狀細(xì)胞(HSCs)在肝纖維化的治療當(dāng)中是關(guān)鍵的靶細(xì)胞,因?yàn)楦卫w維化發(fā)展過(guò)程中細(xì)胞外基質(zhì)(ECM)的沉積主要由活化態(tài)HSCs產(chǎn)生。因此逆轉(zhuǎn)活化態(tài)HSCs的肌成纖維母細(xì)胞表型(myofibroblast,MF)對(duì)肝纖維化和肝炎的治療有著重要的意義。喜樹(shù)堿(CPT)可以抑制腫瘤細(xì)胞中缺氧因子HIF-1-α蛋白的活性;活化態(tài)HSCs細(xì)胞糖酵解代謝活動(dòng)與其MF表型細(xì)胞的增殖緊密相關(guān),本文通過(guò)實(shí)驗(yàn)發(fā)現(xiàn)低劑量CPT可以通過(guò)抑制HIF-1-α蛋白的表達(dá)從而抑制HSCs糖酵解代謝活動(dòng),進(jìn)而抑制其活化態(tài)MF表型,有望成為一種新型治療肝纖維化的藥物。然而CPT難溶于水且其穩(wěn)定性極差,作為裸藥使用時(shí)極其容易被降解和清除,且降解產(chǎn)物毒性很大,因此在本論文中,本文構(gòu)建一種視黃醇(Retinol,VA)偶聯(lián)的兩親性嵌段聚合物材料,并制備成相應(yīng)的膠束,對(duì)CPT進(jìn)行物理包裹后在體內(nèi)載送至肝臟并選擇性靶向HSCs,以期抑制和改善小鼠肝纖維化從而獲得一種新型治療肝纖維化疾病的溫和而有效的藥物。首先,本文合成偶聯(lián)有靶向配體視黃醇的VA-PEG-PCL聚合物并制備成相應(yīng)的納米膠束,未接枝靶向配體的PEG-PCL聚合物設(shè)為對(duì)照。隨后通過(guò)溶劑揮發(fā)法制備VA-PEG-PCL和PEG-PCL納米膠束。通過(guò)核磁共振氫譜(1HNMR)、傅里葉變換紅外光譜(FT-IR)、動(dòng)態(tài)光散射儀(DLS)以及透射電鏡(TEM)對(duì)兩種聚合物材料和自組裝形成的納米膠束理化性質(zhì)進(jìn)行研究,通過(guò)細(xì)胞毒性分析和溶血率測(cè)定來(lái)表征兩種膠束的細(xì)胞相容性和血液相容性,用合成的聚合物制備載CPT膠束并對(duì)載藥膠束進(jìn)行粒徑和體外藥物釋放行為研究。結(jié)果表明,合成的聚合物結(jié)構(gòu)符合預(yù)期;制備的納米膠束粒徑較小且分布較窄,形態(tài)為規(guī)整球形;臨界膠束濃度較小,使其能夠在血液循環(huán)中保持穩(wěn)定的膠束結(jié)構(gòu);兩種膠束均具有良好的細(xì)胞和血液相容性;載CPT納米膠束的藥物釋放性能穩(wěn)定且具有一定的pH響應(yīng)性。體外實(shí)驗(yàn)中,本文研究載有尼羅紅(NileRed,NR)熒光分子的VA-PEG-PCL和PEG-PCL膠束在活化態(tài)HSC-T6細(xì)胞系、原代活化態(tài)HSCs和原代HCs中的細(xì)胞攝取行為,并采用一次性注射四氯化碳(CCl4)誘導(dǎo)急性肝損傷模型研究?jī)煞N膠束所載送的NR在小鼠體內(nèi)主要器官內(nèi)分布。實(shí)驗(yàn)結(jié)果顯示,與PEG-PCL膠束相比,活化態(tài)HSC-T6細(xì)胞系和原代活化態(tài)HSCs細(xì)胞對(duì)偶聯(lián)VA的納米膠束攝取顯著增強(qiáng)。更重要的是,VA-PEG-PCL膠束在進(jìn)入急性肝損傷小鼠體內(nèi)后,較PEG-PCL膠束,大量聚集在受損肝部,表明VA-PEG-PCL納米膠束不僅能夠在體外選擇性靶向HSCs,同樣可選擇性靶向肝纖維化小鼠肝臟中大量擴(kuò)增的HSCs,進(jìn)而把所載送的藥物向肝纖維化小鼠肝臟進(jìn)行靶向性輸送和富集。通過(guò)實(shí)驗(yàn)考察CPT藥物對(duì)活化態(tài)HSCs細(xì)胞存活率、糖酵解活動(dòng)和MF表型的影響,并進(jìn)一步研究CPT裸藥和載CPT膠束對(duì)原代HSCs糖酵解行為和MF表型的抑制效果。實(shí)驗(yàn)結(jié)果表明CPT裸藥對(duì)HSC-T6細(xì)胞生長(zhǎng)、糖酵解行為和MF表型具有抑制作用,且隨著藥物濃度的提高而逐漸增強(qiáng);載CPT膠束對(duì)原代活化態(tài)HSCs基因水平的抑制作用也呈相同趨勢(shì),但因載藥膠束的緩釋作用抑制能力較CPT裸藥降低,同時(shí)由于VA介導(dǎo)受體內(nèi)吞作用,VA-PEG-PCL膠束較PEG-PCL膠束顯示更強(qiáng)的抑制效果。本文采用CCl4腹腔注射六周誘導(dǎo)小鼠慢性肝纖維化模型分析CPT裸藥及載CPT膠束對(duì)肝纖維化小鼠肝臟的治療效果。從生化水平、病理組織學(xué)、分子生物學(xué)進(jìn)行檢測(cè)和分析。實(shí)驗(yàn)結(jié)果表明,模型組小鼠肝臟組織中糖酵解代謝途徑、MF表型標(biāo)記基因、纖維化進(jìn)程相關(guān)基因和蛋白水平顯著增強(qiáng),血清中酶活水平顯著升高,組織石蠟切片染色顯示,模型組小鼠肝臟組織結(jié)構(gòu)存在較嚴(yán)重的破壞,存在較大程度的纖維組織增生間隔肝臟實(shí)質(zhì)結(jié)構(gòu),提示造模成功。CPT裸藥和PEG-PCL載CPT納米膠束組較模型組相關(guān)基因和蛋白水平?jīng)]有明顯恢復(fù),組織切片中纖維化結(jié)構(gòu)特征幾乎未有改善。僅有注射VA-PEG-PCL載送CPT藥物組,能夠保護(hù)CPT并定向輸送至MF表型HSCs細(xì)胞,通過(guò)抑制肝臟組織中的HIF-1-α蛋白表達(dá)進(jìn)而顯著抑制其糖酵解代謝途徑,逆轉(zhuǎn)MF表型并顯著抑制其異常增殖,減少膠原纖維沉積,顯著減輕組織中纖維化結(jié)構(gòu),同時(shí)降低血清中酶活水平并恢復(fù)肝臟的正常功能。采用CCl4誘導(dǎo)小鼠慢性肝纖維化模型探索本課題組前期合成材料葉酸靶向FA-PEG-PCL膠束載CPT對(duì)肝纖維化病變的治療效果。實(shí)驗(yàn)結(jié)果顯示,FA-PEG-PCL載CPT藥物能夠通過(guò)抑制HIF-1-α蛋白水平一定程度抑制病變肝臟中活化態(tài)HSCs糖酵解行為及異常增殖,一定程度減少膠原蛋白的分泌和細(xì)胞外基質(zhì)的沉積,從而改善肝纖維化病變程度同時(shí)部分修復(fù)肝臟功能。
[Abstract]:The activation of hepatic stellate cells (HSCs) in the treatment of liver fibrosis is the key target cells, because the cells of hepatic fibrosis and extracellular matrix (ECM) deposition mainly produced by activated HSCs. Therefore reverse activated HSCs muscle phenotype of fibroblasts (myofibroblast, MF) has important significance the treatment of hepatic fibrosis and hepatitis. Camptothecin (CPT) can inhibit the tumor cell hypoxia factor HIF-1- alpha protein activity in activated HSCs cells; glycolytic activity and phenotype of MF cell proliferation is closely related, we find that low dose of CPT can inhibit HSCs glycolysis activity by inhibiting expression of HIF-1- alpha protein, inhibit the activation state of MF phenotype, is expected to become a new drug for the treatment of liver fibrosis. However, CPT is difficult to dissolve in water and its stability is poor, as naked drug use is easy to be degraded and extremely Clear, and the degradation product is very toxic, so in this paper, we construct a retinol (Retinol, VA) coupling the two amphiphilic diblock polymer materials, and prepared into corresponding micelles, the physical package of CPT in vivo after delivery to the liver and the selective targeting of HSCs, in order to curb and the improvement of hepatic fibrosis in mice so as to obtain a new treatment for liver fibrosis disease mild and effective drug. First of all, this paper is targeted synthesis of VA-PEG-PCL polymer with retinol and prepared into nano micelles of ungrafted polymer ligands targeting PEG-PCL as the control group. Then prepared by solvent evaporation method and VA-PEG-PCL PEG-PCL nanomicelle. By nuclear magnetic resonance spectroscopy (1HNMR), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and transmission electron microscopy (TEM) of two kinds of polymer materials and self-assembly micelles The physicochemical properties were studied by cell toxicity analysis and the hemolysis rate was measured to characterize two kinds of micelle cell compatibility and blood compatibility, polymer synthesis and preparation of CPT loaded micelles on the micelle particle size and in vitro drug release behavior research. The results showed that the synthesis of polymer structure in line with expectations; preparation the micelles with smaller particle size and narrower distribution, morphology is spherical; critical micelle concentration is smaller, the micelle structure can keep stable in blood circulation; two kinds of micelles have good cell and blood compatibility; drug loaded CPT micelles release stable performance and has a certain pH response. In vitro experiments, this paper studies with Nile red (NileRed, NR) VA-PEG-PCL and PEG-PCL fluorescent molecular micelles in activated HSC-T6 cells, primary activation of cellular uptake behavior states of HSCs and HCs in primary, and With a one-time injection of carbon tetrachloride (CCl4) induced acute liver injury model of two kinds of micelles carried by the NR distribution in the main organs of mice. The experimental results show that compared with PEG-PCL micelles, activated HSC-T6 cell lines and primary activated HSCs cell uptake of VA micelles was significantly enhanced. It is more important VA-PEG-PCL, micelles in mice after acute liver injury in PEG-PCL, compared with micelles, gathered in the damaged liver, showed that VA-PEG-PCL micelles can not only to HSCs in vitro selective target, also can selectively target the liver of mice liver fiber amplification of HSCs, and then the carrying drugs targeting liver fibrosis in mice the liver to transport and enrichment. The effects of CPT drugs on activated HSCs cell survival rate by experiment, effects of glycolytic activity and phenotype of MF, and further study the naked CPT drug and loaded CPT glue The inhibitory effect of HSCs on primary beam glycolysis behavior and phenotype of MF. Experimental results show that the naked drug CPT on HSC-T6 cell growth, glycolysis behavior and phenotype of MF has inhibitory effect, and with the increase of drug concentration increased; inhibition of HSCs gene expression on the activation state of the primary load of CPT micelles was also the same trend, but the sustained release of the drug loaded micelles inhibition ability than the naked drug CPT reduced, at the same time as VA mediated by endocytosis in vivo, VA-PEG-PCL micelles of PEG-PCL micelles showed stronger inhibitory effect. This paper uses the CCl4 intraperitoneal injection for six weeks to induce chronic hepatic fibrosis mouse model analysis CPT naked drug and treatment effect of CPT loaded micelles on mice with liver fibrosis. Liver histopathology from biochemical level, molecular biology, detection and analysis. The experimental results show that the liver tissue of model group mice in fermentation solution of metabolic pathways, MF marker gene, fiber The dimension of process related genes and protein levels increased significantly, serum enzyme levels were significantly increased, paraffin section staining showed that the liver tissue of mice model structure exists serious damage, there is a substantial structure of fibrous tissue hyperplasia liver interval greatly, suggesting that the model successfully.CPT naked drugs and PEG-PCL loaded CPT micelles group the model group related genes and protein levels did not significantly improve fibrosis recovery, almost no structural features in tissue sections. The only injection of VA-PEG-PCL carrying CPT drug group, can protect CPT and directional transport to MF phenotype of HSCs cells, and then inhibit the glycolysis pathway by inhibiting HIF-1- protein in liver tissue expression, reverse MF the phenotype and inhibit their proliferation, reduce collagen deposition, reduce fibrosis structure, while a decrease in serum enzyme level and the recovery of liver The normal function of dirty. Using CCl4 induced chronic hepatic fibrosis mice model to explore ourprevious composite folate targeted FA-PEG-PCL CPT loaded micelles on hepatic fibrosis treatment. Experimental results show that FA-PEG-PCL loaded CPT drug that can inhibit glycolysis activation state of HSCs liver lesions in solution behavior and abnormal proliferation by inhibiting HIF-1- protein level to a certain extent, reduce the extent of secretion and deposition of extracellular matrix collagen, so as to improve the degree of liver fibrosis and repair liver function.

【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R943;R965

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