可細胞內(nèi)降解的硬脂酰多肽聚合物基因載體的構(gòu)建及體內(nèi)外評價
發(fā)布時間：2019-06-14 01:42
【摘要】：基因治療(gene therapy)是指將外源基因(RNA或pDNA)導入到特定細胞中,以糾正因基因異�；蜓a償因基因缺陷而引起的各種異常,發(fā)揮治療疾病的作用。目前來看,基因治療技術(shù)在臨床上還不夠成熟,尚未能夠應用于臨床。外源基因片段不能主動進入細胞,需要載體的輔助才能進入細胞發(fā)揮作用,基因治療應用于臨床面臨的一個最大障礙就是缺乏合適的基因載體(gene vector),高效和安全是基因載體的兩項基本要求。因此開發(fā)新型基因載體具有非常重要的意義。本課題基于細胞穿透肽跨膜轉(zhuǎn)運的原理,結(jié)合相關(guān)研究報道,設(shè)計了一種可降解多肽基因載體SHRss,該載體由硬脂酰化的含精氨酸、組氨酸、半胱氨酸多肽經(jīng)二硫鍵橋連而成。體內(nèi)外評價結(jié)果表明,載體具有較高的siRNA遞送效率,同時保持了較低的細胞毒性。本課題的第一部分利用多肽固相合成法合成了多肽H3CR5C,并以同樣的方法將硬脂�；鶊F連接到多肽上得到stearyl-H3CR5C,利用制備高效液相對其進行了純化,HPLC法檢測多肽和硬脂酰多肽的純度在95%以上,HPLC-MS對多肽和硬脂酰多肽進行了鑒定。利用低濃度過氧化氫氧化法,將硬脂酰多肽中的巰基氧化成二硫鍵將其橋連起來得到SHRss,并用半胱氨酸控制橋連程度,H-NMR和GPC結(jié)果表明SHRss成功合成。本課題的第二部分對可降解多肽基因載體SHRss進行了體外評價。SHRss/siRNA納米復合物在N/P=10時的粒徑最小,粒徑在150-300nm之間,電位在25-40mV之間,透射電鏡觀察到的納米復合物近似球形,結(jié)構(gòu)完整。SHRss2在N/P=5可以完全包裹住siRNA,并增加siRNA在血清中的穩(wěn)定性。Luc-Hela細胞對SHRss/siRNA納米復合物具有有非常高的細胞攝取。SHRss/si RNA納米復合物在Luc-Hela細胞和mCherry-HEK293細胞中均表現(xiàn)出較高的siRNA遞送效率。入胞機制研究結(jié)果表明,SHRss/siRNA納米復合物的入胞途徑主要為網(wǎng)格蛋白介導的內(nèi)吞和質(zhì)膜微囊介導的內(nèi)吞。激光共聚焦顯微鏡觀察發(fā)現(xiàn),SHRss/Cy3-siRNA納米復合物在入胞后可以成功的從內(nèi)涵體逃逸,均勻的分布于細胞質(zhì)。SHRss的細胞毒性小于Lipofectamine2000和PEI 25kDa,具有良好的生物相容性,二硫鍵的引入沒有明顯增加載體的毒性。本課題的第三部分對四種可降解多肽基因載體在體外評價中表現(xiàn)最佳的SHRss2進行了荷瘤裸鼠遞送siRNA的體內(nèi)研究。結(jié)果表明SHRss2/si RNA納米復合物在尾靜脈注射后可以較多的累積在荷瘤裸鼠的腫瘤組織中。SHRss2/si RNA納米復合物對腫瘤組織中相關(guān)基因的表達有比較強干擾作用。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R450
本文編號：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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R450
【參考文獻】
相關(guān)期刊論文 前1條
1 王良友,潘和平,陳正英;多肽合成中幾種形成二硫鍵方法的介紹[J];有機化學;1998年06期
，本文編號：2498992
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