可细胞内降解的硬脂酰多肽聚合物基因载体的构建及体内外评价
发布时间：2019-06-14 01:42
【摘要】：基因治疗(gene therapy)是指将外源基因(RNA或pDNA)导入到特定细胞中,以纠正因基因异常或补偿因基因缺陷而引起的各种异常,发挥治疗疾病的作用。目前来看,基因治疗技术在临床上还不够成熟,尚未能够应用于临床。外源基因片段不能主动进入细胞,需要载体的辅助才能进入细胞发挥作用,基因治疗应用于临床面临的一个最大障碍就是缺乏合适的基因载体(gene vector),高效和安全是基因载体的两项基本要求。因此开发新型基因载体具有非常重要的意义。本课题基于细胞穿透肽跨膜转运的原理,结合相关研究报道,设计了一种可降解多肽基因载体SHRss,该载体由硬脂酰化的含精氨酸、组氨酸、半胱氨酸多肽经二硫键桥连而成。体内外评价结果表明,载体具有较高的siRNA递送效率,同时保持了较低的细胞毒性。本课题的第一部分利用多肽固相合成法合成了多肽H3CR5C,并以同样的方法将硬脂酰基团连接到多肽上得到stearyl-H3CR5C,利用制备高效液相对其进行了纯化,HPLC法检测多肽和硬脂酰多肽的纯度在95%以上,HPLC-MS对多肽和硬脂酰多肽进行了鉴定。利用低浓度过氧化氢氧化法,将硬脂酰多肽中的巯基氧化成二硫键将其桥连起来得到SHRss,并用半胱氨酸控制桥连程度,H-NMR和GPC结果表明SHRss成功合成。本课题的第二部分对可降解多肽基因载体SHRss进行了体外评价。SHRss/siRNA纳米复合物在N/P=10时的粒径最小,粒径在150-300nm之间,电位在25-40mV之间,透射电镜观察到的纳米复合物近似球形,结构完整。SHRss2在N/P=5可以完全包裹住siRNA,并增加siRNA在血清中的稳定性。Luc-Hela细胞对SHRss/siRNA纳米复合物具有有非常高的细胞摄取。SHRss/si RNA纳米复合物在Luc-Hela细胞和mCherry-HEK293细胞中均表现出较高的siRNA递送效率。入胞机制研究结果表明,SHRss/siRNA纳米复合物的入胞途径主要为网格蛋白介导的内吞和质膜微囊介导的内吞。激光共聚焦显微镜观察发现,SHRss/Cy3-siRNA纳米复合物在入胞后可以成功的从内涵体逃逸,均匀的分布于细胞质。SHRss的细胞毒性小于Lipofectamine2000和PEI 25kDa,具有良好的生物相容性,二硫键的引入没有明显增加载体的毒性。本课题的第三部分对四种可降解多肽基因载体在体外评价中表现最佳的SHRss2进行了荷瘤裸鼠递送siRNA的体内研究。结果表明SHRss2/si RNA纳米复合物在尾静脉注射后可以较多的累积在荷瘤裸鼠的肿瘤组织中。SHRss2/si RNA纳米复合物对肿瘤组织中相关基因的表达有比较强干扰作用。
[Abstract]:Gene therapy (gene therapy) refers to the introduction of foreign genes (RNA or pDNA) into specific cells to correct all kinds of abnormalities caused by gene abnormalities or compensate for gene defects, and to play an important role in the treatment of diseases. At present, gene therapy technology is not mature enough in clinic and has not yet been applied in clinic. Foreign gene fragments can not enter the cells actively and need the assistance of vectors to enter the cells to play a role. one of the biggest obstacles to the clinical application of gene therapy is that the lack of suitable gene vector (gene vector), is the two basic requirements of gene vectors. Therefore, it is of great significance to develop new gene vectors. Based on the principle of cell penetrating peptide transmembrane transport and related research reports, a biodegradable polypeptide gene vector SHRss, was designed, which was composed of stearyl acylated arginine, histidine and cysteine polypeptides bridged by disulfide bonds. The results of in vitro and in vivo evaluation showed that the carrier had high efficiency of siRNA delivery and maintained low cytotoxicity. In the first part of this paper, the polypeptide H _ 3CR5C was synthesized by solid phase synthesis of polypeptide, and the stearyl group was connected to the polypeptide by the same method to obtain stearyl-H3CR5C,. The purity of polypeptide and stearyl polypeptide was over 95% by HPLC. The polypeptide and stearyl polypeptide were identified by HPLC-MS. The sulfhydryl group in stearyl polypeptide was oxidized to disulfide bond by low concentration hydrogen peroxide oxidation to obtain SHRss, and the degree of bridge was controlled by cystein. the results of H-NMR and GPC showed that SHRss was synthesized successfully. In the second part of this study, the biodegradable polypeptide gene vector SHRss was evaluated in vitro. the particle size of HRSS / siRNA nanocomposites in N/P=10 was the smallest, the particle size was between 150-300nm and the potential was between 25-40mV. The nanocomposites observed by transmission electron microscope were approximately spherical, and the structure of HRSS / HRss2 could be completely wrapped in siRNA, in N/P=5. Luc-Hela cells had a very high cellular uptake of SHRss/siRNA nanocomposites. HRSS / siRNA nanocomposites showed high siRNA delivery efficiency in Luc-Hela cells and mCherry-HEK293 cells, and increased the stability of HRSS in serum. Luc-Hela cells had a very high cellular uptake of HRSS / HRSS nanocomposites. HRSS / HRSS nanocomposites showed high siRNA delivery efficiency in Luc-Hela cells and mCherry-HEK293 cells. The results showed that the entrapment pathway of SHRss/siRNA nanocomposites was mainly endocytosis mediated by grid protein and endocytosis mediated by plasma membrane microcapsules. The results of laser confocal microscope showed that SHRss/Cy3-siRNA nanocomposites could escape from the endosome successfully after entering the cell. the cytotoxicity of HRSS uniformly distributed in cytoplasm was less than that of Lipofectamine2000 and PEI 25kDa. it had good biocompatibility. The introduction of disulfide bond did not significantly increase the toxicity of the carrier. In the third part of this study, four biodegradable polypeptide gene vectors, SHRss2, which performed the best performance in vitro, were studied in vivo to deliver siRNA to tumor-bearing nude mice. The results showed that SHRss2/si RNA nanocomposites could accumulate in tumor tissues of nude mice bearing tumor after tail vein injection. SHRss2/si RNA nanocomposites strongly interfered with the expression of related genes in tumor tissues.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450
本文编号：2498992
[Abstract]:Gene therapy (gene therapy) refers to the introduction of foreign genes (RNA or pDNA) into specific cells to correct all kinds of abnormalities caused by gene abnormalities or compensate for gene defects, and to play an important role in the treatment of diseases. At present, gene therapy technology is not mature enough in clinic and has not yet been applied in clinic. Foreign gene fragments can not enter the cells actively and need the assistance of vectors to enter the cells to play a role. one of the biggest obstacles to the clinical application of gene therapy is that the lack of suitable gene vector (gene vector), is the two basic requirements of gene vectors. Therefore, it is of great significance to develop new gene vectors. Based on the principle of cell penetrating peptide transmembrane transport and related research reports, a biodegradable polypeptide gene vector SHRss, was designed, which was composed of stearyl acylated arginine, histidine and cysteine polypeptides bridged by disulfide bonds. The results of in vitro and in vivo evaluation showed that the carrier had high efficiency of siRNA delivery and maintained low cytotoxicity. In the first part of this paper, the polypeptide H _ 3CR5C was synthesized by solid phase synthesis of polypeptide, and the stearyl group was connected to the polypeptide by the same method to obtain stearyl-H3CR5C,. The purity of polypeptide and stearyl polypeptide was over 95% by HPLC. The polypeptide and stearyl polypeptide were identified by HPLC-MS. The sulfhydryl group in stearyl polypeptide was oxidized to disulfide bond by low concentration hydrogen peroxide oxidation to obtain SHRss, and the degree of bridge was controlled by cystein. the results of H-NMR and GPC showed that SHRss was synthesized successfully. In the second part of this study, the biodegradable polypeptide gene vector SHRss was evaluated in vitro. the particle size of HRSS / siRNA nanocomposites in N/P=10 was the smallest, the particle size was between 150-300nm and the potential was between 25-40mV. The nanocomposites observed by transmission electron microscope were approximately spherical, and the structure of HRSS / HRss2 could be completely wrapped in siRNA, in N/P=5. Luc-Hela cells had a very high cellular uptake of SHRss/siRNA nanocomposites. HRSS / siRNA nanocomposites showed high siRNA delivery efficiency in Luc-Hela cells and mCherry-HEK293 cells, and increased the stability of HRSS in serum. Luc-Hela cells had a very high cellular uptake of HRSS / HRSS nanocomposites. HRSS / HRSS nanocomposites showed high siRNA delivery efficiency in Luc-Hela cells and mCherry-HEK293 cells. The results showed that the entrapment pathway of SHRss/siRNA nanocomposites was mainly endocytosis mediated by grid protein and endocytosis mediated by plasma membrane microcapsules. The results of laser confocal microscope showed that SHRss/Cy3-siRNA nanocomposites could escape from the endosome successfully after entering the cell. the cytotoxicity of HRSS uniformly distributed in cytoplasm was less than that of Lipofectamine2000 and PEI 25kDa. it had good biocompatibility. The introduction of disulfide bond did not significantly increase the toxicity of the carrier. In the third part of this study, four biodegradable polypeptide gene vectors, SHRss2, which performed the best performance in vitro, were studied in vivo to deliver siRNA to tumor-bearing nude mice. The results showed that SHRss2/si RNA nanocomposites could accumulate in tumor tissues of nude mice bearing tumor after tail vein injection. SHRss2/si RNA nanocomposites strongly interfered with the expression of related genes in tumor tissues.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450
【参考文献】
相关期刊论文 前1条
1 王良友,潘和平,陈正英;多肽合成中几种形成二硫键方法的介绍[J];有机化学;1998年06期
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