自组装siRNA-DNA纳米管构建及其调节肺动脉平滑肌细胞自噬作用的研究

发布时间：2018-05-25 07:39

本文选题：DNA纳米结构 + 自组装　；参考：《第三军医大学》2015年硕士论文

【摘要】：基因治疗在疾病治疗中具有巨大的应用潜力,研究可以传递各种目的基因的载体是纳米医学最重要的研究课题之一。目前,已经报道了许多药物传递载体,如高分子聚合物、阳离子脂质体、各种纳米材料、病毒衣壳等。这些传递系统的有效性已被证实,然而其大部分为外源性物质,缺乏组织特异性且具有潜在毒性。随着DNA分子自组装纳米技术的发展,自组装DNA纳米结构在生物学领域的应用变成现实。DNA分子的优势在于其稳定性,易生物降解,与其它的药物传递材料相比,DNA分子对于人体具有天然性、无免疫原性、无毒等优点。大量研究显示,自组装DNA纳米结构可以用于细胞内的货物传递。并且,DNA纳米结构可以依据需递送的信号分子、缓释需要或者靶向配体等因素进行结构的优化。因此,自组装DNA纳米材料是理想的递送平台。肺动脉高压(Pulmonary arterial hypertension,PAH)严重威胁人类健康,患者5年生存率只有60%。低氧诱导肺动脉平滑肌细胞(Pulmonary arterial smooth muscle cells,PASMCs)异常增殖和凋亡抵抗导致的肺血管重构(Pulmonary vascular remodeling,PVR)是肺动脉高压发生的重要病理生理基础。自噬是一种广泛存在于哺乳动物细胞中的保守的、维持细胞内稳态的机制。最近的研究证实,其在肺血管疾病,如肺动脉高压、慢性阻塞性肺疾病的发生、发展中发挥着重要作用。然而,自噬在肺动脉高压血管重构过程中,特别是在肺动脉平滑肌细胞生物学效应的影响尚不十分清楚。故深入研究自噬在低氧诱导PASMCs增殖中的作用与机制,从而进一步阐明低氧致肺血管重构的分子机制,可望为防治PAH提供新的靶点及策略。鉴于此,我们设计研发了一种以四条DNA单链自组装的DNA纳米管,并结合DNA-RNA杂交技术将功能基因mTOR siRNA以特定的比例组装进入纳米管结构。研究分析其有效性,并深入探讨其对常氧或低氧条件下大鼠PASMCs自噬与增殖的影响。研究结论将进一步完善低氧致肺血管重构的分子机制,有望为肺高压等血管重构性疾病的防治提供新的理论依据和策略。目的:自组装携带mtorsirna的dna纳米管,分析其有效性,并研究其对rpasmcs自噬和增殖的影响。方法:1.自组装携带mtorsirna的dna纳米管(sirna-nts)及其特性研究⑴采用序列对称性原理,三臂星形模块方案自组装携带mtorsirna的dna纳米管;⑵通过凝胶电泳,原子力显微镜分析、表征dna纳米管的结构;2.携带mtorsirna的dna纳米管在rpasmcs的摄取及转染效率观察体外培养来源于sd大鼠的肺动脉平滑肌细胞(rpasmcs),由sirna-nts转染rpasmcs;应用激光共聚焦显微镜观察rpasmcs对不同浓度的sirna-nts的摄取以及同一浓度不同时间点的摄取情况;流式细胞术检测sirna-nts在rpasmcs的转染效率及细胞内摄取平均荧光强度;通过激光共聚焦观察dna纳米管在rpasmcs的内吞进程。3.携带mtorsirna的dna纳米管对常氧下rpasmcs自噬作用的研究由sirna-nts转染rpasmcs,激光共聚焦显微镜观察sirna-nts对rpasmcs自噬的影响;透射电镜对比观察sirna-nts诱导rpasmcs自噬的细胞超微结构;rt-pcr检测sirna-nts转染rpasmcs后mtormrna的表达;westernblot检测sirna-nts转染rpasmcs后,p-mtor、t-mtor、lc3b和pacn的表达;mtt法检测sirna-nts对rpasmcs生长抑制作用的浓度及时间依赖性。4.携带mtorsirna的dna纳米管对低氧下rpasmcs自噬作用的研究由sirna-nts转染rpasmcs,低氧处理。激光共聚焦显微镜观察sirna-nts对低氧下rpasmcs自噬的影响;westernblot检测sirna-nts转染rpasmcs后,p-mtor、lc3b和pacn的表达变化;mtt、3h-tdr检测sirna-nts转染rpasmcs后,对细胞活力及增殖的影响。结果:1.纳米材料的构建和表征分析。(1)成功自组装携带mtorsirna的dna纳米管(sirna-nts);(2)非变性page胶分析、表征其产率高、结构稳定;(3)原子力显微镜扫描显示,dna纳米管粒子粒度分布均匀。2.细胞摄取携带mtorsirna的dna纳米管具有剂量及时间依赖的特点。(1)激光共聚焦显微镜观察细胞摄取sirnacy3-nts具有剂量及时间依赖性,随剂量及转染孵育时间的增加,细胞内红色荧光颗粒逐渐增多,且当浓度为50nm及孵育12-24h达到较高的细胞内摄取(p0.05)。并显著强于单独的sirna对照组(p0.05)(2)流式细胞术检测结果显示细胞摄取sirnacy3-nts具有剂量及时间依赖性,随剂量及转染孵育时间的增加,细胞转染效率逐渐升高,且当浓度为50nm及转染孵育12-24h达到较高的转染效率(p0.05)。同时,细胞内摄取的平均荧光密度也具有剂量及时间依赖性,且显著强于单独的sirna组(p0.05)。上述结果提示自组装的sirna-dna纳米管结构可以有效促进其携带的mtorsirna转运进入rpasmcs内,同时稳定释放。(3)激光共聚焦观察sirnacy3-nts纳米粒子在细胞的内吞进程,结果显示cy3红色荧光标记的携带mtorsirna的dna纳米管随时间延长逐渐从胞膜进入胞质,并定位于内涵体。3.携带mtorsirna的dna纳米管系统影响肺动脉平滑肌细胞自噬和增殖。(1)激光共聚焦图像显示红色荧光标记的sirnacy3-nts与绿色标记的细胞内自噬小体位于同一区域。(2)mtorsirna诱导肺动脉平滑肌细胞自噬。激光共聚焦图像显示sirna-nts组细胞内可见大量标记自噬小体的绿色荧光,且显著强于对照组及单独的sirna组(p0.05),甚至明显强于雷帕霉素组(阳性对照组)(p0.05)。透射电镜图像显示:sirna-nts组细胞内可见大量自噬小体聚集,明显多于对照组及单独的纳米管及sirna组。westernblot结果及半定量分析显示sirna-nts组lc3bii/i蛋白表达显著强于单独的sirna组及单独的纳米管组(p0.05)。(3)携带mtorsirna的dna纳米管通过抑制mtor信号增强lc3b蛋白表达。rt-pcr结果显示mtormrna表达抑制具有剂量及时间依赖性。且当浓度为50nm及转染孵育48h达到较高的抑制率(p0.05)。westernblot结果显示p-mtor及t-mtor蛋白表达抑制具有时间依赖性,孵育48h、72h的mtor蛋白水平明显低于24h(p0.05)。westernblot结果显示lc3bii/i蛋白表达随mtor蛋白表达抑制而升高,且于48h达到最高峰,相反,pcna蛋白表达随mtor蛋白表达抑制而下降。提示通过下调mtor表达,诱导细胞自噬,抑制增殖。(4)mtt检测结果显示:携带mtorsirna的dna纳米管以浓度及时间依赖的方式抑制细胞生长。4.mtor信号调节低氧诱导的细胞自噬和增殖。(1)携带mtorsirna的dna纳米管促进低氧诱导的细胞自噬。激光共聚焦图像显示sirna-nts组细胞内可见大量标记自噬小体的绿色荧光,且显著强于低氧对照组及单独sirna组。westernblot结果显示低氧组lc3bii/i蛋白表达较常氧组升高(p0.05),sirna-nts组lc3bii/i蛋白表达明显强于其它各组(p0.05)。(2)携带mtorsirna的dna纳米管抑制低氧诱导的细胞增殖。mtt结果显示:sirna-nts组细胞活力明显下降,且显著低于低氧下其它各组(P0.05)。[3H]Td R结果显示:si RNA-NTs组细胞增殖较低氧对照组明显下降,且明显低于单独的siRNA组(P0.05)。(3)mTOR调节低氧诱导的LC3B及PCNA蛋白表达。Westernblot结果显示siRNA-NTs组抑制p-mTOR及PCNA蛋白表达,同时升高LC3B蛋白表达水平。提示抑制mTOR表达,可以诱导细胞自噬,导致细胞自噬性死亡,进而抑制细胞增殖。结论:1.成功自组装携带mTOR siRNA的DNA纳米管,该纳米管粒子结构稳定,通过内吞途径进入细胞,其细胞转染和摄取呈时间和剂量依赖性。2.携带mTOR si RNA的DNA纳米管显著抑制常氧和低氧下肺动脉平滑肌细胞中m TOR的表达,进而诱导细胞自噬,抑制细胞增殖。
[Abstract]:Gene therapy has great potential in the treatment of disease. It is one of the most important research topics to study the delivery of various target genes. At present, many drug delivery carriers, such as polymers, cationic liposomes, various nanomaterials, virus capsid, etc. have been reported. These transmission systems are effective. Sex has been confirmed, but most of it is exogenous, lack of tissue specificity and potential toxicity. With the development of DNA molecular self-assembled nanotechnology, the advantage of self assembled DNA nanostructures in the field of biology into realistic.DNA molecules is its stability, biodegradation, and other drug delivery materials, DNA A large number of studies have shown that self-assembled DNA nanostructures can be used for the delivery of goods in cells. Moreover, DNA nanostructures can be transmitted according to the required signaling molecules, and the structure is optimized by slow release needs or targeted ligands. Therefore, self assembly of DNA nanomaterials is made. It is an ideal delivery platform. Pulmonary arterial hypertension (PAH) is a serious threat to human health. The 5 year survival rate of patients is only 60%. hypoxic induced pulmonary artery smooth muscle cells (Pulmonary arterial smooth muscle cells, PASMCs) abnormal proliferation and apoptosis resistance caused by pulmonary vascular remodeling. It is an important pathophysiological basis for the occurrence of pulmonary arterial hypertension. Autophagy is a conservative mechanism that exists widely in mammalian cells and maintains intracellular homeostasis. Recent studies have shown that it plays an important role in the development of pulmonary vascular diseases, such as pulmonary hypertension, chronic obstructive pulmonary disease, and autophagy in the lung. In the process of vascular remodeling, especially in the biological effects of pulmonary artery smooth muscle cells, the effects and mechanisms of autophagy in hypoxia induced PASMCs proliferation are studied. The molecular mechanism of hypoxia induced pulmonary vascular remodeling is further elucidated, and the new targets and strategies for the prevention and control of PAH are expected. In view of this, We designed and developed a self-assembled DNA nanotube with four DNA single stranded chains, and assembled the functional gene mTOR siRNA into the nanotube structure in a specific proportion combined with DNA-RNA hybridization. The effect of the mTOR siRNA on the autophagy and proliferation of PASMCs in normoxic or hypoxic rats was investigated and analyzed. To improve the molecular mechanism of hypoxia induced pulmonary vascular remodeling, it is expected to provide a new theoretical basis and strategy for the prevention and control of vascular remodeling diseases such as pulmonary hypertension. Objective: self-assembled mtorsirna DNA nanotubes, analyze its effectiveness, and study its effect on rpasmcs autophagy and proliferation. Method: 1. self-assembled DNA nanotubes (SIR) carrying mtorsirna (SIR) Na-nts) and its characteristics study (1) using the principle of sequence symmetry, the three arm star module scheme self assembled the DNA nanotubes carrying mtorsirna; (2) the structure of the DNA nanotubes was characterized by gel electrophoresis and atomic force microscopy; 2. the uptake and transfection efficiency of mtorsirna loaded DNA nanotubes in rpasmcs were derived from SD rats in vitro Pulmonary artery smooth muscle cells (rpasmcs) were transfected to rpasmcs by sirna-nts; the uptake of sirna-nts at different concentrations by rpasmcs and the uptake of the same concentration at different time points were observed by laser confocal microscopy; flow cytometry was used to detect the transfection efficiency of sirna-nts in rpasmcs and the average fluorescence intensity of intracellular uptake; the laser copolymerization was made by laser copolymerization. Focal observation of DNA nanotubes in rpasmcs endocytosis process.3. carrying mtorsirna DNA nanotube to rpasmcs autophagy under normal oxygen by sirna-nts transfected rpasmcs, laser confocal microscope observation of the effect of sirna-nts on rpasmcs autophagy; transmission electron microscopy compared the ultrastructure of sirna-nts induced rpasmcs autophagy. The expression of mtormrna after transfection of rpasmcs with na-nts; Westernblot detection of the expression of p-mTOR, t-mtor, lc3b and pacn after sirna-nts transfection of rpasmcs; MTT method for detecting the concentration and time dependence of sirna-nts on the growth inhibition of sirna-nts Treatment. The effect of sirna-nts on rpasmcs autophagy under hypoxia; Westernblot detection of p-mTOR, lc3b and pacn expression changes after sirna-nts transfection of rpasmcs; MTT, 3H-TdR test sirna-nts transfection rpasmcs, the effect on cell viability and proliferation. Results: 1. nanomaterials construction and characterization analysis. (1) successful self-assembly DNA nanotube (sirna-nts) carrying mtorsirna, (2) non denatured PAGE gel analysis, characterized by high yield and stable structure; (3) atomic force microscopy showed that the particle size distribution of DNA nanotube particles was distributed uniformly in.2. cells carrying the DNA nanotubes carrying mtorsirna in a time dependent manner. (1) laser confocal microscopy observed cell uptake of Si Rnacy3-nts had dose and time dependence. With the increase of dose and incubation time, the intracellular red fluorescent particles increased gradually, and when the concentration was 50nm and incubated 12-24h reached a higher intracellular uptake (P0.05). And significantly stronger than the separate siRNA control group (P0.05) (2) flow cytometry results showed that the cell uptake of sirnacy3-nts With the dose and time dependence, the transfection efficiency increased gradually with the increase of dose and transfection time, and high transfection efficiency (P0.05) was achieved when the concentration was 50nm and the transfection was incubated with 12-24h (P0.05). Meanwhile, the average fluorescence density of the intracellular uptake was also dependent on the dose and time, and was significantly stronger than the single siRNA group (P0.05). The results suggest that self-assembled sirna-dna nanotube structure can effectively promote the transport of mtorsirna transport into rpasmcs and stabilize release. (3) laser confocal observation of the endocytosis of sirnacy3-nts nanoparticles in cells shows that the Cy3 red fluorescent labeled DNA nanotubes with mtorsirna are gradually extending from the cell with time. The membrane enters the cytoplasm and determines the autophagy and proliferation of the pulmonary artery smooth muscle cells by the DNA nanotube system at the endosomal.3. carrying mtorsirna. (1) the laser confocal image shows that the red fluorescent labeled sirnacy3-nts and the green labeled autophagosomes are located in the same region. (2) mtorsirna induces autophagy in the pulmonary artery smooth muscle cells. The confocal imaging showed that the green fluorescence of a large number of autophagic corpuscles was found in the sirna-nts group, and significantly stronger than the control group and the separate siRNA group (P0.05), and even significantly stronger than the rapamycin group (P0.05). The transmission electron microscope images showed that a large number of autophagic corpuscles were found in the sirna-nts group, obviously more than the control group and the control group. .westernblot results and semi quantitative analysis of the single nanotube and siRNA group showed that the expression of lc3bii/i protein in sirna-nts group was significantly stronger than that of the single siRNA group and the single nanotube group (P0.05). (3) the DNA nanotubes carrying mtorsirna enhanced the.Rt-pcr result of the lc3b protein expression by inhibiting the mTOR signal and showed that the mtormrna expression inhibition was in a dose and timely manner. Inter dependence, and when the concentration of 50nm and transfected 48h reached a higher inhibitory rate (P0.05).Westernblot results showed that the inhibition of p-mTOR and t-mtor protein expression was time dependent, the mTOR protein level of 72h was significantly lower than 24h (P0.05).Westernblot results showed that the expression of proteins increased with the inhibition of protein expression. 48h reached the peak, on the contrary, the expression of PCNA protein decreased with the inhibition of the expression of mTOR protein. It suggested that the expression of mTOR could be downregulated to induce autophagy and inhibit proliferation. (4) the results of MTT detection showed that the DNA nanotubes carrying mtorsirna in a concentration and time dependent manner inhibited the cell autophagy induced by the long.4.mtor signal and the regulation of hypoxia induced autophagy and proliferation. (1) the DNA nanotubes carrying mtorsirna promoted autophagy induced by hypoxia. The laser confocal image showed that the green fluorescence of a large number of autophagic bodies in the sirna-nts group was visible, and significantly stronger than the hypoxia control group and the single siRNA group.Westernblot results showed that the lc3bii/ I protein expression in the hypoxia group was higher than that of the normal oxygen group (P0.05), sirna-nts (P0.05). The expression of lc3bii/i protein in the group was significantly stronger than that in other groups (P0.05). (2) the DNA nanotubes carrying mtorsirna inhibited the proliferation of hypoxia induced cell proliferation.Mtt results showed that the cell viability of sirna-nts group decreased significantly, and was significantly lower than that of other groups under hypoxia (P0.05).[3H]Td R results: Si RNA-NTs group cell proliferation was significantly lower than the hypoxia control group. Significantly lower than the single siRNA group (P0.05). (3) mTOR regulated hypoxia induced LC3B and PCNA protein expression.Westernblot results showed that the siRNA-NTs group inhibited the expression of p-mTOR and PCNA protein, and increased the expression level of LC3B protein. It suggested that the inhibition of mTOR expression could induce autophagy and induce autophagic death, and then inhibit cell proliferation. 1. successfully self assembled DNA nanotubes carrying mTOR siRNA, the nanotube particles are stable and enter cells through endocytic pathway. The transfection and uptake of DNA nanotubes with time and dose dependent.2. carrying mTOR Si RNA significantly inhibit the expression of M TOR in the fine cells of pulmonary artery smooth muscle under normooxy and hypoxia, and then induce autophagy to inhibit cell autophagy. Cell proliferation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R450;TB383.1

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