本文選題：DNA納米結(jié)構(gòu) + 自組裝　； 參考：《第三軍醫(yī)大學》2015年碩士論文

【摘要】：基因治療在疾病治療中具有巨大的應用潛力,研究可以傳遞各種目的基因的載體是納米醫(yī)學最重要的研究課題之一。目前,已經(jīng)報道了許多藥物傳遞載體,如高分子聚合物、陽離子脂質(zhì)體、各種納米材料、病毒衣殼等。這些傳遞系統(tǒng)的有效性已被證實,然而其大部分為外源性物質(zhì),缺乏組織特異性且具有潛在毒性。隨著DNA分子自組裝納米技術(shù)的發(fā)展,自組裝DNA納米結(jié)構(gòu)在生物學領(lǐng)域的應用變成現(xiàn)實。DNA分子的優(yōu)勢在于其穩(wěn)定性,易生物降解,與其它的藥物傳遞材料相比,DNA分子對于人體具有天然性、無免疫原性、無毒等優(yōu)點。大量研究顯示,自組裝DNA納米結(jié)構(gòu)可以用于細胞內(nèi)的貨物傳遞。并且,DNA納米結(jié)構(gòu)可以依據(jù)需遞送的信號分子、緩釋需要或者靶向配體等因素進行結(jié)構(gòu)的優(yōu)化。因此,自組裝DNA納米材料是理想的遞送平臺。肺動脈高壓(Pulmonary arterial hypertension,PAH)嚴重威脅人類健康,患者5年生存率只有60%。低氧誘導肺動脈平滑肌細胞(Pulmonary arterial smooth muscle cells,PASMCs)異常增殖和凋亡抵抗導致的肺血管重構(gòu)(Pulmonary vascular remodeling,PVR)是肺動脈高壓發(fā)生的重要病理生理基礎。自噬是一種廣泛存在于哺乳動物細胞中的保守的、維持細胞內(nèi)穩(wěn)態(tài)的機制。最近的研究證實,其在肺血管疾病,如肺動脈高壓、慢性阻塞性肺疾病的發(fā)生、發(fā)展中發(fā)揮著重要作用。然而,自噬在肺動脈高壓血管重構(gòu)過程中,特別是在肺動脈平滑肌細胞生物學效應的影響尚不十分清楚。故深入研究自噬在低氧誘導PASMCs增殖中的作用與機制,從而進一步闡明低氧致肺血管重構(gòu)的分子機制,可望為防治PAH提供新的靶點及策略。鑒于此,我們設計研發(fā)了一種以四條DNA單鏈自組裝的DNA納米管,并結(jié)合DNA-RNA雜交技術(shù)將功能基因mTOR siRNA以特定的比例組裝進入納米管結(jié)構(gòu)。研究分析其有效性,并深入探討其對常氧或低氧條件下大鼠PASMCs自噬與增殖的影響。研究結(jié)論將進一步完善低氧致肺血管重構(gòu)的分子機制,有望為肺高壓等血管重構(gòu)性疾病的防治提供新的理論依據(jù)和策略。目的:自組裝攜帶mtorsirna的dna納米管,分析其有效性,并研究其對rpasmcs自噬和增殖的影響。方法:1.自組裝攜帶mtorsirna的dna納米管(sirna-nts)及其特性研究⑴采用序列對稱性原理,三臂星形模塊方案自組裝攜帶mtorsirna的dna納米管;⑵通過凝膠電泳,原子力顯微鏡分析、表征dna納米管的結(jié)構(gòu);2.攜帶mtorsirna的dna納米管在rpasmcs的攝取及轉(zhuǎn)染效率觀察體外培養(yǎng)來源于sd大鼠的肺動脈平滑肌細胞(rpasmcs),由sirna-nts轉(zhuǎn)染rpasmcs;應用激光共聚焦顯微鏡觀察rpasmcs對不同濃度的sirna-nts的攝取以及同一濃度不同時間點的攝取情況;流式細胞術(shù)檢測sirna-nts在rpasmcs的轉(zhuǎn)染效率及細胞內(nèi)攝取平均熒光強度;通過激光共聚焦觀察dna納米管在rpasmcs的內(nèi)吞進程。3.攜帶mtorsirna的dna納米管對常氧下rpasmcs自噬作用的研究由sirna-nts轉(zhuǎn)染rpasmcs,激光共聚焦顯微鏡觀察sirna-nts對rpasmcs自噬的影響;透射電鏡對比觀察sirna-nts誘導rpasmcs自噬的細胞超微結(jié)構(gòu);rt-pcr檢測sirna-nts轉(zhuǎn)染rpasmcs后mtormrna的表達;westernblot檢測sirna-nts轉(zhuǎn)染rpasmcs后,p-mtor、t-mtor、lc3b和pacn的表達;mtt法檢測sirna-nts對rpasmcs生長抑制作用的濃度及時間依賴性。4.攜帶mtorsirna的dna納米管對低氧下rpasmcs自噬作用的研究由sirna-nts轉(zhuǎn)染rpasmcs,低氧處理。激光共聚焦顯微鏡觀察sirna-nts對低氧下rpasmcs自噬的影響;westernblot檢測sirna-nts轉(zhuǎn)染rpasmcs后,p-mtor、lc3b和pacn的表達變化;mtt、3h-tdr檢測sirna-nts轉(zhuǎn)染rpasmcs后,對細胞活力及增殖的影響。結(jié)果:1.納米材料的構(gòu)建和表征分析。(1)成功自組裝攜帶mtorsirna的dna納米管(sirna-nts);(2)非變性page膠分析、表征其產(chǎn)率高、結(jié)構(gòu)穩(wěn)定;(3)原子力顯微鏡掃描顯示,dna納米管粒子粒度分布均勻。2.細胞攝取攜帶mtorsirna的dna納米管具有劑量及時間依賴的特點。(1)激光共聚焦顯微鏡觀察細胞攝取sirnacy3-nts具有劑量及時間依賴性,隨劑量及轉(zhuǎn)染孵育時間的增加,細胞內(nèi)紅色熒光顆粒逐漸增多,且當濃度為50nm及孵育12-24h達到較高的細胞內(nèi)攝取(p0.05)。并顯著強于單獨的sirna對照組(p0.05)(2)流式細胞術(shù)檢測結(jié)果顯示細胞攝取sirnacy3-nts具有劑量及時間依賴性,隨劑量及轉(zhuǎn)染孵育時間的增加,細胞轉(zhuǎn)染效率逐漸升高,且當濃度為50nm及轉(zhuǎn)染孵育12-24h達到較高的轉(zhuǎn)染效率(p0.05)。同時,細胞內(nèi)攝取的平均熒光密度也具有劑量及時間依賴性,且顯著強于單獨的sirna組(p0.05)。上述結(jié)果提示自組裝的sirna-dna納米管結(jié)構(gòu)可以有效促進其攜帶的mtorsirna轉(zhuǎn)運進入rpasmcs內(nèi),同時穩(wěn)定釋放。(3)激光共聚焦觀察sirnacy3-nts納米粒子在細胞的內(nèi)吞進程,結(jié)果顯示cy3紅色熒光標記的攜帶mtorsirna的dna納米管隨時間延長逐漸從胞膜進入胞質(zhì),并定位于內(nèi)涵體。3.攜帶mtorsirna的dna納米管系統(tǒng)影響肺動脈平滑肌細胞自噬和增殖。(1)激光共聚焦圖像顯示紅色熒光標記的sirnacy3-nts與綠色標記的細胞內(nèi)自噬小體位于同一區(qū)域。(2)mtorsirna誘導肺動脈平滑肌細胞自噬。激光共聚焦圖像顯示sirna-nts組細胞內(nèi)可見大量標記自噬小體的綠色熒光,且顯著強于對照組及單獨的sirna組(p0.05),甚至明顯強于雷帕霉素組(陽性對照組)(p0.05)。透射電鏡圖像顯示:sirna-nts組細胞內(nèi)可見大量自噬小體聚集,明顯多于對照組及單獨的納米管及sirna組。westernblot結(jié)果及半定量分析顯示sirna-nts組lc3bii/i蛋白表達顯著強于單獨的sirna組及單獨的納米管組(p0.05)。(3)攜帶mtorsirna的dna納米管通過抑制mtor信號增強lc3b蛋白表達。rt-pcr結(jié)果顯示mtormrna表達抑制具有劑量及時間依賴性。且當濃度為50nm及轉(zhuǎn)染孵育48h達到較高的抑制率(p0.05)。westernblot結(jié)果顯示p-mtor及t-mtor蛋白表達抑制具有時間依賴性,孵育48h、72h的mtor蛋白水平明顯低于24h(p0.05)。westernblot結(jié)果顯示lc3bii/i蛋白表達隨mtor蛋白表達抑制而升高,且于48h達到最高峰,相反,pcna蛋白表達隨mtor蛋白表達抑制而下降。提示通過下調(diào)mtor表達,誘導細胞自噬,抑制增殖。(4)mtt檢測結(jié)果顯示:攜帶mtorsirna的dna納米管以濃度及時間依賴的方式抑制細胞生長。4.mtor信號調(diào)節(jié)低氧誘導的細胞自噬和增殖。(1)攜帶mtorsirna的dna納米管促進低氧誘導的細胞自噬。激光共聚焦圖像顯示sirna-nts組細胞內(nèi)可見大量標記自噬小體的綠色熒光,且顯著強于低氧對照組及單獨sirna組。westernblot結(jié)果顯示低氧組lc3bii/i蛋白表達較常氧組升高(p0.05),sirna-nts組lc3bii/i蛋白表達明顯強于其它各組(p0.05)。(2)攜帶mtorsirna的dna納米管抑制低氧誘導的細胞增殖。mtt結(jié)果顯示:sirna-nts組細胞活力明顯下降,且顯著低于低氧下其它各組(P0.05)。[3H]Td R結(jié)果顯示:si RNA-NTs組細胞增殖較低氧對照組明顯下降,且明顯低于單獨的siRNA組(P0.05)。(3)mTOR調(diào)節(jié)低氧誘導的LC3B及PCNA蛋白表達。Westernblot結(jié)果顯示siRNA-NTs組抑制p-mTOR及PCNA蛋白表達,同時升高LC3B蛋白表達水平。提示抑制mTOR表達,可以誘導細胞自噬,導致細胞自噬性死亡,進而抑制細胞增殖。結(jié)論:1.成功自組裝攜帶mTOR siRNA的DNA納米管,該納米管粒子結(jié)構(gòu)穩(wěn)定,通過內(nèi)吞途徑進入細胞,其細胞轉(zhuǎn)染和攝取呈時間和劑量依賴性。2.攜帶mTOR si RNA的DNA納米管顯著抑制常氧和低氧下肺動脈平滑肌細胞中m TOR的表達,進而誘導細胞自噬,抑制細胞增殖。
[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.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R450;TB383.1

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