PSMA适配子—穿膜肽—siRNA靶向高效递送系统的建立及其功能验证

发布时间：2018-03-14 17:55

本文选题：前列腺癌　切入点：靶向治疗　出处：《第四军医大学》2016年博士论文　论文类型：学位论文

【摘要】：内容:目的:尽管RNA干扰技术已经被开发为前列腺癌治疗的新方法,但是体内应用时靶向特异地将治疗性siRNA递送至目标肿瘤组织仍然存在很大挑战。迄今为止关于siRNA递送的研究关注于改善递送靶向性和将siRNA跨膜递送至胞内,但是两者是两个相对独立的研究方向[39]。联合应用特异递送以及跨膜递送策略无疑较单一策略具有优势,但联合策略目前尚未见报道。此项研究的目的是通过递送策略的联合应用构建PMSA适配子-穿膜肽-siRNA靶向高效递送系统A10-STD-(sursiRNA),该递送复合体系旨在同时解决siRNA递送的特异性和高效性两个问题。方法:治疗性siRNA分子(sur-siRNA)针对survivin基因降低其表达发挥抑瘤作用。为改善递送特异性,选择适配子A10介导sur-siRNA靶向递送;为改善递送高效性,选择穿膜肽TAT-DRBD介导sur-siRNA跨膜;为了偶联两种策略,选择链霉亲和素SA-生物素系统进行偶联。首先用原核表达体系表达融合蛋白STD,发挥蛋白骨架功能。STD由链霉亲和素SA(S)、穿膜肽TAT(T)和双链RNA结合蛋白DRBD(D)三部分组成。然后STD通过SA与生物素标记的抗前列腺癌特异性膜抗原(PSMA)的适配子A10结合,以及通过DRBD与抗survivin基因的siRNA(sur-siRNA)结合。递送复合体建立后通过与无转染策略以及单一递送策略对照组进行比较,进一步在递送的靶向性、高效性以及抑瘤效能方面进行功能验证。结果:利用大肠杆菌原核表达系统(pET44b质粒/Rosetta-gami菌株)成功表达了SA-3TAT-DRBD(STD)融合蛋白。经验证通过两步低温条件下STD融合蛋白与A10及siRNA共孵育递送复合体A10-STD-(sur-siRNA)组装完成。经验证该递送复合体系能特异性靶向PSMA阳性的前列腺癌细胞或者组织,并能将有效地将sur-siRNA递送至LNCaP细胞系的胞质中。与其他常用siRNA递送试剂脂质体2000以及单纯靶向策略A10-(sur-siRNA)嵌合体比较,分别提高19.2%及59.9%的递送效率以及16.8%及26.1%的促细胞凋亡率。体内实验证明经尾静脉注射sur-siRNA,递送复合体组较其他两组能明显抑制肿瘤组织生长(p0.001)。结论:治疗性siRNA的靶向高效递送系统A10-STD-(sur-siRNA)能特异性及高效性地将治疗性sur-siRNA递送至靶向肿瘤细胞,在体内应用时能有效抑制肿瘤生长,证明联合递送策略的有效性。该靶向高效递送系统作为一种siRNA递送的新型策略在前列腺癌siRNA治疗方面显示出广阔的应用前景。
[Abstract]:Content: objective: although RNA interference technology has been developed as a new therapy for prostate cancer, However, there is still a great challenge in targeting specific delivery of therapeutic siRNA to target tumor tissues in vivo. So far, research on siRNA delivery has focused on improving delivery targeting and transmembrane delivery of siRNA into cells. But they are two relatively independent research directions [39]. There is no doubt that the combined application of specific delivery and transmembrane delivery strategies has advantages over a single strategy. However, the joint strategy has not been reported yet. The aim of this study is to construct PMSA aptamer-transmembrane peptide-siRNA targeting high efficiency delivery system A10-STD-sursiRNAs, which is designed to solve the special problems of siRNA delivery at the same time. Methods: therapeutic siRNA molecule sur-siRNAs play a role in inhibiting the expression of survivin gene in order to improve the delivery specificity. Select aptamer A10 to mediate sur-siRNA targeted delivery, to improve delivery efficiency, select transmembrane peptide TAT-DRBD to mediate sur-siRNA transmembrane, and to couple two strategies, Firstly, the fusion protein STD was expressed in prokaryotic expression system, and the protein skeleton function. STD was composed of three parts: streptavidin (SAG), transmembrane peptide (TATT) and double stranded RNA binding protein (DRBDX). After STD binding to the aptamer A10, a biotinylated antigen-specific membrane antigen against prostate cancer, was mediated by SA. After the establishment of the delivery complex, the delivery complex was compared with the control group of non-transfection strategy and single delivery strategy, and the targeting of the delivery was further enhanced by the combination of DRBD with siRNA-siRNA-siRNA-siRNA-siRNA-siRNA-siRNA-siRNA-siRNA-resistant to survivin gene. Results: using E. coli prokaryotic expression system pET44b plasmid / Rosetta-gami strain, we successfully expressed SA-3TAT-DRBDD-STD) fusion protein. It was verified that SA-3TAT-DRBDD-STD fusion protein and A10 fusion protein were obtained under two-step hypothermia condition by using E. coli prokaryotic expression system (pET44b plasmid / Rosetta-gami strain). And siRNA co-incubated delivery complex A10-STD-sur-siRNAs were assembled. It was proved that the delivery system could specifically target PSMA positive prostate cancer cells or tissues. Compared with other common siRNA delivery reagent liposome 2000 and targeting strategy A10-Osur-siRNAs, the chimerism of sur-siRNA could be effectively transferred to the cytoplasm of LNCaP cell line. The delivery efficiency of 19.2% and 59.9% and the apoptotic rate of 16.8% and 26.1% were increased respectively. In vivo, it was proved that sur-siRNA-delivery complex group could significantly inhibit tumor tissue growth compared with the other two groups. Conclusion: the target of therapeutic siRNA can be treated with sur-siRNAs. A10-STD-sur-siRNAs, a highly efficient delivery system, can specifically and efficiently deliver therapeutic sur-siRNA to targeted tumor cells. It can effectively inhibit tumor growth in vivo and prove the effectiveness of the combined delivery strategy. As a novel siRNA delivery strategy, the targeted high-efficiency delivery system has a broad application prospect in the treatment of prostate cancer siRNA.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25

【参考文献】