本文選題：前列腺癌　切入點：靶向治療　出處：《第四軍醫(yī)大學》2016年博士論文　論文類型：學位論文

【摘要】：內容:目的:盡管RNA干擾技術已經被開發(fā)為前列腺癌治療的新方法,但是體內應用時靶向特異地將治療性siRNA遞送至目標腫瘤組織仍然存在很大挑戰(zhàn)。迄今為止關于siRNA遞送的研究關注于改善遞送靶向性和將siRNA跨膜遞送至胞內,但是兩者是兩個相對獨立的研究方向[39]。聯(lián)合應用特異遞送以及跨膜遞送策略無疑較單一策略具有優(yōu)勢,但聯(lián)合策略目前尚未見報道。此項研究的目的是通過遞送策略的聯(lián)合應用構建PMSA適配子-穿膜肽-siRNA靶向高效遞送系統(tǒng)A10-STD-(sursiRNA),該遞送復合體系旨在同時解決siRNA遞送的特異性和高效性兩個問題。方法:治療性siRNA分子(sur-siRNA)針對survivin基因降低其表達發(fā)揮抑瘤作用。為改善遞送特異性,選擇適配子A10介導sur-siRNA靶向遞送;為改善遞送高效性,選擇穿膜肽TAT-DRBD介導sur-siRNA跨膜;為了偶聯(lián)兩種策略,選擇鏈霉親和素SA-生物素系統(tǒng)進行偶聯(lián)。首先用原核表達體系表達融合蛋白STD,發(fā)揮蛋白骨架功能。STD由鏈霉親和素SA(S)、穿膜肽TAT(T)和雙鏈RNA結合蛋白DRBD(D)三部分組成。然后STD通過SA與生物素標記的抗前列腺癌特異性膜抗原(PSMA)的適配子A10結合,以及通過DRBD與抗survivin基因的siRNA(sur-siRNA)結合。遞送復合體建立后通過與無轉染策略以及單一遞送策略對照組進行比較,進一步在遞送的靶向性、高效性以及抑瘤效能方面進行功能驗證。結果:利用大腸桿菌原核表達系統(tǒng)(pET44b質粒/Rosetta-gami菌株)成功表達了SA-3TAT-DRBD(STD)融合蛋白。經驗證通過兩步低溫條件下STD融合蛋白與A10及siRNA共孵育遞送復合體A10-STD-(sur-siRNA)組裝完成。經驗證該遞送復合體系能特異性靶向PSMA陽性的前列腺癌細胞或者組織,并能將有效地將sur-siRNA遞送至LNCaP細胞系的胞質中。與其他常用siRNA遞送試劑脂質體2000以及單純靶向策略A10-(sur-siRNA)嵌合體比較,分別提高19.2%及59.9%的遞送效率以及16.8%及26.1%的促細胞凋亡率。體內實驗證明經尾靜脈注射sur-siRNA,遞送復合體組較其他兩組能明顯抑制腫瘤組織生長(p0.001)。結論:治療性siRNA的靶向高效遞送系統(tǒng)A10-STD-(sur-siRNA)能特異性及高效性地將治療性sur-siRNA遞送至靶向腫瘤細胞,在體內應用時能有效抑制腫瘤生長,證明聯(lián)合遞送策略的有效性。該靶向高效遞送系統(tǒng)作為一種siRNA遞送的新型策略在前列腺癌siRNA治療方面顯示出廣闊的應用前景。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R737.25

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本文編號：1612312