基于猪细小病毒VLPs的靶向纳米载体的构建和靶向性研究

发布时间：2018-04-14 13:17

本文选题：纳米载体 + 猪细小病毒　；参考：《黑龙江八一农垦大学》2017年硕士论文

【摘要】：纳米递送系统是纳米医药学的一个重要分支,在提高药物靶向性,减少药物毒副作用及改善治疗效果等方面发挥着重要作用。病毒样颗粒(VLPs)作为新型的纳米载体成为近年来纳米递送系统的研究热点之一。基于猪细小病毒(PPV)结构蛋白VP2可自组装配成病毒样颗粒,本研究通过基因工程技术将其进行改造,以获得具有靶向的纳米载体。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区域扩增TK-vp2基因,在杆状病毒表达系统中构建重组杆粒Bacmid-TK-vp2,转染至昆虫细胞Sf9中,利用直接免疫荧光试验、SDS-PAGE、western-blot以及透射电镜检测TK-VP2蛋白表达和自组装,再用氯化铯(Cs Cl)密度梯度离心技术纯化。通过化学交联方法将TK-VLPs与阿霉素(DOX)或荧光探针FAM共价交联,0.6%琼脂糖凝胶电泳鉴定,利用透射电镜和动态光散射仪对TK-VLPs-DOX外观形态、粒径及粒度分布测定。利用激光共聚焦和流式细胞仪检测Caco-2、HRT-18和HUVEC细胞对TK-VLPs-FAM和TK-VLPs-DOX的摄取情况,用于其评价细胞摄取的体外靶向性。构建皮下HRT-18肿瘤裸鼠模型,尾静脉注射TK-VLPs-FAM,利用小动物活体成像仪和免疫荧光染色方法验证TK-VLPs的体内靶向性。采用MTT法检测TK-VLPs-DOX对Caco-2和HRT-18细胞的体外生长抑制作用。SOE-PCR扩增获得TK-vp2基因,在杆状病毒表达系统中构建获得Bacmid-TK-vp2,转染至昆虫细胞Sf9中,直接免疫荧光试验、SDS-PAGE、Western-blot和透射电镜鉴定成功表达TK-VP2蛋白,自组装成TK-VLPs。Cs Cl密度梯度离心纯化得到TK-VLPs纳米粒子,透射电镜观察证实纯化的TK-VLPs形态呈球形,粒径约为22 nm左右。0.6%琼脂糖凝胶电泳鉴定TK-VLPs与DOX成功共价交联形成TK-VLPs-DOX,透射电镜和动态光散射仪测定证实TK-VLPs-DOX形态呈球形,平均粒径约为28 nm,粒度分布均匀。激光共聚焦显微镜观察和流式细胞仪测定表明TK-VLPs-FAM能被Caco-2、HRT-18和HUVEC细胞摄取,TK-VLPs-DOX能被Caco-2和HRT-18细胞所摄取。小动物活体成像仪和免疫荧光染色鉴定表明TK-VLPs-FAM不仅在肿瘤细胞中有分布而且能与肿瘤新生血管共定位。MTT法测定结果显示TK-VLPs-DOX能够显著抑制Caco-2和HRT-18细胞。本研究成功获得一种新型的纳米载体TK-VLPs,为纳米递药系统实现临床应用奠定了重要基础,也为病毒的基础研究和纳米医药应用提供了新的路径。
[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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.651

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