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非病毒载体质粒DNA已被广泛应用于基因治疗和DNA疫苗,目前迫切需要开发其大规模制备和分离纯化方法。亲和色谱是一种高分辨率、高选择性的分离技术,在蛋白质、抗体、核酸等生物大分子的分离纯化方面显示了良好的应用前景。本文综述了亲和色谱技术在超螺旋质粒DNA分离纯化中的研究进展,总结了各种亲和色谱方法分离超螺旋质粒DNA的机理和优缺点,并展望了亲和纯化技术在质粒DNA生产和制备中的应用前景。 相似文献
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Yasuya Kudo Kazunori Koiwai Kazuhiro Shimizu Shota Kusuki Mina Sakuragi Naohiko Shimada Yoichi Takeda Kazuo Sakurai 《中国化学快报》2008,19(9):1115-1118
A lipoplex (i.e., pDNA#1/lipid complex and transfection reagent for pDNA delivery) containing galactosylceramide (GalCer) and an amidine-bearing lipid (TRX) was examined whether the bound pDNA was specifically ingested by hepatocyte via asialoglycoprotein receptor (ASGPR) and then expressed protein. Gel electrophoresis and small-angle X-ray scattering (SAXS) confirmed that the TRX-GalCer liposome#2 complexed with pDNA and the resultant lipoplex took a hexagonally packed inverted cylinder structure when the GalCer composition was less than 20 wt.% of the total lipid. When the lipoplex carrying pGL3 (luciferase-cording pDNA) was administrated to HepG2, the luciferase activity was increased with increasing the GalCer composition until it reached 3 wt.% and then decreased upon further addition of GalCer. When we added galactose itself as a competitor, the luciferase activity was decreased, while glucose did not show such decrease, suggesting that HepG2 ingested the lipoplex via ASGPR-mediated endocytosis. This paper indicated that the hexagonally packed inverted cylinder structures of lipoplex may not always provide excellent transfection and presented a possibility that the TRX lipoplex#3 can obtain a cellulartargeting ability through the receptors for oligosaccharide. 相似文献
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带有人凝血因子Ⅸ cDNA的反转录病毒载体的构建及其在离体细胞中的高效表达 总被引:1,自引:0,他引:1
报道了带有人凝血因子IX cDNA的高滴度高表达安全性反转录病毒载体的构建。采用LNL6反转录病毒载体为骨架,构建了由人巨细胞病毒启动子(hCMV)驱动的反转录病毒载体LNCIX,由反转录病毒LTR启动子驱动的反转录病毒载体LIXSN,以及由LTR和CMV启动子共同控制转录的反转录病毒载体LCIXSN,分别用电穿孔方法转导PA317辅助细胞株。LNCIX和LIXSN反转录病毒载体能在离体细胞中表达人IX因子蛋白,而LC'IXSN反转录病毒载体转移到离体细胞中没有检测到人IX因子蛋白。PA317/INCIX细胞的产病毒滴度为8×10~5CFU/ml,该重组病毒感染人纤维肉瘤细胞HT1080及血友病B患者皮肤成纤维细胞(HSF),用ELISA方法分别测定这些细胞的IX因子蛋白产量,LNCIX载体在HT-1080细胞中的人IX因子平均表达量为3.3μg/10~6细胞·d~(-1);在HSF细胞中的平均表达量为2.5μg/10~6细胞·d~(-1),其中80%以上的IX因子具有凝血活性,与过去相比,提高了产病毒滴度,增加了人IX因子的平均表达量,病毒载体骨架的设计更完善,降低了产生野生型病毒的概率,提高了安全性,对于进一步开展血友病B的基因治疗具有重要的意义。 相似文献
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同载基因和药物的超微载体粒子的制备及体外评价 总被引:4,自引:0,他引:4
以聚乳酸_乙醇酸共聚物 (PLGA)和自行制备的O_羧甲基壳聚糖 (O_CMC)为原料 ,以 5_氟尿嘧啶 (5 Fu)为抗癌药物模型 ,以反义EGFR(表皮生长因子受体 )为基因药物模型 ,构建与评价了同载抗癌药物与基因的复合功能纳米药物载体系统。同载超微粒子的平均粒径为 2 5 8 7nm ,粒径分布指数为 0 14 2 ,粒子表面 ξ电位为 - 10 6 7eV。同载超微粒子在PBS中的释药行为研究表明 :超微粒子中 5_FU和基因均具有零级缓慢释放特性。体外肿瘤细胞存活率实验和免疫组化实验均证实同载超微粒子能高效抑制TJ90 5人脑胶质瘤细胞的增殖。最后用荧光共聚焦显微镜动态监测了超微粒子进入瘤细胞的转染过程 ,发现粒子可在不同时间内进入细胞浆和细胞核。 相似文献
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基因治疗这种堪称革命性的治疗方法,开拓了治疗癌症的新思路,其最关键性问题是实现核酸药物靶向肿瘤组织并精准治疗。核酸药物直接递送存在核酸酶降解代谢、细胞膜上的负电荷排斥现象以及稳定性差等问题,所以核酸药物需要载体协助,成功的载体递送除能使核酸药物在肿瘤区域大量富集外,还要起到药物控释作用,而天然多糖除无毒、生物相容度高、易修饰的特点外,它本身就具有免疫调节、抗肿瘤、抗炎等多种生物活性。本篇总结了最具代表性的五种多糖的结构特征及在核酸药物递送方面的应用,继而归纳了多糖的常用的纳米级载体形式,为构建天然多糖递送核酸的新型载体并将其应用到免疫抗肿瘤治疗研究中奠定基础。 相似文献
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肿瘤治疗是目前医学研究的重点方向,其方法包括传统的放疗、化疗、手术以及目前热门的各种基因治疗等,各有其优缺点。而p53基因治疗与放射治疗方法的结合越来越受到重视。综述了近年来p53基因转导联合放射治疗恶性肿瘤的研究结果以及可能的作用机理及进展。Cancer treatment is one of the most important fields in medical research. All strategies such as radiotherapy, chemotherapy, surgery, and gene-based therapy have their own advantages and disadvantages. Nowadays, a novel method which combined p53-gene therapy with radiotherapy plays an important role in the field of cancer research. This review summarized the current state of combined therapies of p53-genetherapy and radiotherapy, possible mechanism and recent progress. 相似文献
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目前肿瘤基因治疗尚存在许多问题, 距临床应用还有相当的距离, 但是在传统的放疗、化疗和手术治疗的基础上, 辐射与基因治疗的有机结合在肿瘤治疗中却显示出可喜的前景。综述了近年来这一领域的研究进展, 探讨了这一疗法对肿瘤治疗的应用前景。Although tumor gene therapy has a distance to clinical use due to some problems, the combination of irradiation and gene therapy holds much promise in cancer therapy based on the traditional radiotherapy, chemotherapy and surgery. We have termed this therapeutic radiogenic therapy. This review focuses on the group of radiogenic therapy that are either: ⑴ improvement of gene transfer efficiency by irradiation; ⑵ radiotherapy combined with cytokines gene delivery or enhancement of the immunity of tumor cells by transgene; ⑶ directly stimulated by radiation to produce either directly or indirectly cytotoxic agents; ⑷ increasing of radiosensitivity in gene therapy; ⑸ radioprotective gene therapy enhances radiation tumor killing effect while protecting the normal tissue and organs with transgene using transfer vector. 相似文献