本文選題：納米載體 + 豬細(xì)小病毒��； 參考：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文

【摘要】：納米遞送系統(tǒng)是納米醫(yī)藥學(xué)的一個重要分支,在提高藥物靶向性,減少藥物毒副作用及改善治療效果等方面發(fā)揮著重要作用。病毒樣顆粒(VLPs)作為新型的納米載體成為近年來納米遞送系統(tǒng)的研究熱點之一�；谪i細(xì)小病毒(PPV)結(jié)構(gòu)蛋白VP2可自組裝配成病毒樣顆粒,本研究通過基因工程技術(shù)將其進行改造,以獲得具有靶向的納米載體。采用SOE-PCR方法把具有靶向的TK肽基因序列插入到vp2基因序列的loop2和loop4區(qū)域擴增TK-vp2基因,在桿狀病毒表達系統(tǒng)中構(gòu)建重組桿粒Bacmid-TK-vp2,轉(zhuǎn)染至昆蟲細(xì)胞Sf9中,利用直接免疫熒光試驗、SDS-PAGE、western-blot以及透射電鏡檢測TK-VP2蛋白表達和自組裝,再用氯化銫(Cs Cl)密度梯度離心技術(shù)純化。通過化學(xué)交聯(lián)方法將TK-VLPs與阿霉素(DOX)或熒光探針FAM共價交聯(lián),0.6%瓊脂糖凝膠電泳鑒定,利用透射電鏡和動態(tài)光散射儀對TK-VLPs-DOX外觀形態(tài)、粒徑及粒度分布測定。利用激光共聚焦和流式細(xì)胞儀檢測Caco-2、HRT-18和HUVEC細(xì)胞對TK-VLPs-FAM和TK-VLPs-DOX的攝取情況,用于其評價細(xì)胞攝取的體外靶向性。構(gòu)建皮下HRT-18腫瘤裸鼠模型,尾靜脈注射TK-VLPs-FAM,利用小動物活體成像儀和免疫熒光染色方法驗證TK-VLPs的體內(nèi)靶向性。采用MTT法檢測TK-VLPs-DOX對Caco-2和HRT-18細(xì)胞的體外生長抑制作用。SOE-PCR擴增獲得TK-vp2基因,在桿狀病毒表達系統(tǒng)中構(gòu)建獲得Bacmid-TK-vp2,轉(zhuǎn)染至昆蟲細(xì)胞Sf9中,直接免疫熒光試驗、SDS-PAGE、Western-blot和透射電鏡鑒定成功表達TK-VP2蛋白,自組裝成TK-VLPs。Cs Cl密度梯度離心純化得到TK-VLPs納米粒子,透射電鏡觀察證實純化的TK-VLPs形態(tài)呈球形,粒徑約為22 nm左右。0.6%瓊脂糖凝膠電泳鑒定TK-VLPs與DOX成功共價交聯(lián)形成TK-VLPs-DOX,透射電鏡和動態(tài)光散射儀測定證實TK-VLPs-DOX形態(tài)呈球形,平均粒徑約為28 nm,粒度分布均勻。激光共聚焦顯微鏡觀察和流式細(xì)胞儀測定表明TK-VLPs-FAM能被Caco-2、HRT-18和HUVEC細(xì)胞攝取,TK-VLPs-DOX能被Caco-2和HRT-18細(xì)胞所攝取。小動物活體成像儀和免疫熒光染色鑒定表明TK-VLPs-FAM不僅在腫瘤細(xì)胞中有分布而且能與腫瘤新生血管共定位。MTT法測定結(jié)果顯示TK-VLPs-DOX能夠顯著抑制Caco-2和HRT-18細(xì)胞。本研究成功獲得一種新型的納米載體TK-VLPs,為納米遞藥系統(tǒng)實現(xiàn)臨床應(yīng)用奠定了重要基礎(chǔ),也為病毒的基礎(chǔ)研究和納米醫(yī)藥應(yīng)用提供了新的路徑。
[Abstract]:Nano-delivery system is an important branch of nano-medicine, which plays an important role in improving drug targeting, reducing side effects and improving therapeutic effect.As a new type of nano-carrier, virus-like particles (VLPs) have become one of the research hotspots in nanodelivery systems in recent years.Based on the self-assembly of porcine parvovirus (PPV) structural protein VP2 to form virus-like particles, the novel nanoparticles were modified by genetic engineering to obtain targeted nanoparticles.The target TK peptide gene sequence was inserted into the loop2 and loop4 region of the vp2 gene sequence by SOE-PCR method to amplify the TK-vp2 gene. The recombinant baculoid mid-BacTK-vp2 was constructed in the baculovirus expression system and transfected into the insect cell Sf9.The expression and self-assembly of TK-VP2 protein were detected by SDS-PAGEX western-blot and transmission electron microscopy, and then purified by Cs chloride density gradient centrifugation.The covalent crosslinking of TK-VLPs with doxorubicin (DOX) or fluorescent probe FAM (0.6% agarose gel electrophoresis) was used to determine the morphology, particle size and particle size distribution of TK-VLPs-DOX by transmission electron microscopy (TEM) and dynamic light scattering (DLS).The uptake of TK-VLPs-FAM and TK-VLPs-DOX in Caco-2G HRT-18 and HUVEC cells was detected by confocal laser and flow cytometry.A nude mouse model of subcutaneous HRT-18 tumor was established and TK-VLPs-FAM was injected into the tail vein. The in vivo targeting of TK-VLPs was verified by small animal in vivo imager and immunofluorescence staining.The inhibitory effect of TK-VLPs-DOX on the growth of Caco-2 and HRT-18 cells in vitro was detected by MTT assay. The TK-vp2 gene was amplified by SOE-PCR. Bacmid-TK-vp2 was constructed in baculovirus expression system and transfected into insect Sf9.TK-VP2 protein was successfully expressed by SDS-PAGEG Western-blot and transmission electron microscopy (TEM). The TK-VLPs nanoparticles were purified by self-assembled TK-VLPs.Cs Cl density gradient centrifugation. The morphology of the purified TK-VLPs was spherical by transmission electron microscopy (TEM).The particle size of TK-VLPs-DOX was about 22 nm. 0.6% agarose gel electrophoresis showed that TK-VLPs and DOX were covalent crosslinked to form TK-VLPs-DOX. The morphology of TK-VLPs-DOX was spherical by transmission electron microscopy and dynamic light scattering. The average diameter of TK-VLPs-DOX was about 28 nm, and the particle size distribution was uniform.Laser confocal microscopy and flow cytometry showed that TK-VLPs-FAM could be ingested by Caco-2OHRT-18 and HUVEC cells and TK-VLPs-DOX by Caco-2 and HRT-18 cells.TK-VLPs-FAM not only distributed in tumor cells but also co-located with tumor neovascularization. The results showed that TK-VLPs-DOX could significantly inhibit Caco-2 and HRT-18 cells.In this study, a novel nano-carrier TK-VLPswas successfully obtained, which laid an important foundation for the clinical application of nano-delivery system, and also provided a new path for the basic research of virus and the application of nano-medicine.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.651

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本文編號：1749480