心肌靶向纳米粒的构建及其功能效应的初步评价

发布时间：2018-01-24 22:48

本文关键词： 脓毒症心肌损伤纳米粒心肌靶向　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景脓毒症(sepsis)是由细菌、真菌、病毒等感染因素导致的全身性炎症反应,起病急骤、病情危重、机制复杂、预后较差是其重要特征,通常会引起全身多脏器功能障碍、衰竭,已成为危重患者的常见死亡原因之一。研究表明,25%以上的脓毒症患者合并心血管并发症,心肌损伤是脓毒症患者预后不良的重要标志。目前临床上并无针对性的措施来减轻心肌损伤,因此如何保护心功能,对改善危重患者身体状况、提高脓毒症的救治率具有重要意义。NF-κB(nuclear factor-kappa B)是当代研究的热点,在过度炎症反应发生过程中起关键性作用。研究证实,脓毒症发生时,NF-κB的活性明显升高,此时应用siRNA(Small interfering Ribonucleic Acid)抑制NF-κB的P65亚基可有效抑制炎症反应。传统的给药方式中siRNA在体内易被降解,纳米技术不仅能有效保护siRNA,也能通过化学修饰后获得主动靶向功能。β_1-肾上腺素受体(β_1-Adrenergic Receptors,β_1-AR)在人体内主要分布于心脏,心肌损伤时,膜表面受体的数量与敏感性均显著上调,可作为纳米粒的效应靶点。亲脂性化合物3-{4-[2-羟基-(1-甲基乙胺基)丙氧基]苯基}丙酸十六醇酯(PAC)与β_1-AR受体选择性阻滞剂艾司洛尔(esmolol)具有类似的化学结构,以往的研究表明PAC修饰的脂质体具有较强的心肌细胞特异靶向性。故本课题设想,利用RNA干扰技术以及复合纳米材料的靶向性,体外构建β_1-AR介导的P65-NF-κB-siRNA心肌细胞靶向纳米粒,并通过尾静脉注射导入小鼠体内,观察其在小鼠体内的分布情况,建立急性内毒素心肌损伤模型,研究其对心肌细胞的保护作用,从而实现减轻脓毒症心肌损伤、保护心肌功能、提高脓毒症救治率的目的。研究方法1.艾司洛尔类似物PAC的合成通过查阅文献及总结交流,确定了以3-(4-羟基苯基)丙酸为起始原料,依次经过羟基与十六烷醇成酯、环氧丙基化、异丙胺化等步骤,最后得到终产物PAC的合成路线。2.siRNA的设计合成与筛选针对P65基因,设计合成3种不同的siRNA及阴性对照siRNA,利用lipo2000将siRNA分别转染到小鼠心肌细胞系(HL-1)细胞内,培养24h后提取RNA行PCR扩增,检测各组基因表达情况,筛选出干扰效果最好的siRNA。3.纳米粒的合成通过查阅文献和借鉴经验,确定了以siRNA+聚乙二醇(polyethylene glycol,PEG)为内核、以聚乳酸-羟基乙酸共聚物[Poly(lactic-co-glycolic acid),PLGA]为纳米材料、以维生素E聚乙二醇琥珀酸酯(Vitamin E-Tocopheryl Polyethylene Glycol 1000Succinate,TPGS)为乳化剂的制作方法,优化试剂配比及操作步骤,制备心肌靶向纳米粒,并利用激光粒径仪、Zeta电位分析仪、扫描电子显微镜等进行各表征分析,通过紫外分光光度计检测并计算纳米粒的包封率、载药量、释放率等一系列指标。通过单因素分析优化制备工艺,以得到性质最佳的纳米粒。4.纳米粒的细胞实验体外培养大鼠心肌细胞系(HL-1),荧光显微镜检测细胞表面β_1-AR受体表达情况。将各组纳米粒加入细胞培养混悬液中,通过荧光显微镜和流式细胞学技术检测细胞对纳米粒的摄取及siRNA在细胞内的分布情况。将si NC纳米粒和si435纳米粒分别转染细胞,培养24h后提取RNA进行PCR扩增,明确基因表达情况。5.纳米粒的动物实验将不同纳米粒通过尾静脉注射到C57BL/6J小鼠体内,24h后小鼠腹腔注射LPS(10mg/Kg)制作脓毒症损伤模型,小动物成像仪检测纳米粒在体内的分布情况;心尖穿刺取血检测心肌酶谱、炎症因子等的变化,取心脏组织行凋亡基因检测和组织切片染色以明确心肌损伤情况。研究结果1.合成的艾司洛尔类似物PAC纯度高,性质稳定,无生物学毒性,可用于进一步的合成研究。2.转染si435的细胞中m RNA水平表达最低,说明si435对NF-κB的P65亚基抑制作用最强,最终选择si435作为心肌靶向纳米粒的核心。3.经过不同配比的合成分析比对,875ul 5%PEG+125ul siRNA作为内水相,80mg PLGA+15mg TPGS+5mg PAC溶于8ml丙酮作为有机相,100ml 0.03%TPGS作为外水相,按照此配比合成的纳米粒粒径均一,分散性好,电势分布集中,此时的包封率,载药量,释放曲线也比较稳定。4.小鼠心肌细胞系(HL-1)细胞膜表面有明显β_1-AR受体表达;不同浓度的空白纳米粒与细胞共培养,细胞存活率未受影响,说明纳米粒对细胞无毒性;荧光显微镜和流式细胞学检测纳米粒在细胞内的分布情况,发现纳米粒分布均匀,细胞摄取率高,能稳定进入细胞内部并产生明显的P65抑制效应。5.心肌靶向纳米粒均有较强的心肌细胞靶向性,说明纳米粒能靶向识别心肌细胞;小鼠腹腔注射LPS(10mg/Kg)可导致明显的心肌损伤,而注射si435纳米粒组的心肌损伤程度明显减轻;脓毒症发生时,CK、LDH、HBDH均明显升高,纳米粒保护组的表达则显著降低;炎症因子、凋亡相关基因的表达情况也均有同样的趋势,说明si435纳米粒能有效保护心肌细胞、降低脓毒症心肌损伤。研究结论1.成功合成艾司洛尔类似物PAC,纯度高,性质稳定,无生物学毒性,可用于进一步的合成研究。2.筛选出干扰效果最好的siRNA—si435,能明显抑制NF-κB P65亚基的表达。3.成功合成了PAC-P65-NF-Κb-siRNA心肌靶向纳米粒,性质稳定,粒径均一,包封率高,载药性能好,释放曲线稳定。4.纳米粒对细胞无生物学毒性,其包裹的siRNA能释放到心肌细胞内并产生干扰效应,减轻炎症反应。5.纳米粒能显著改善心肌组织的脓毒症损伤程度,减轻炎症反应,从而保护心脏功能。
[Abstract]:Backgroundsepsis (sepsis) is caused by bacteria, fungi, systemic inflammation, virus infection factors lead to abrupt onset, in critical condition, complex mechanism, poor prognosis is one of the most important characteristics, usually cause multiple organ dysfunction and failure, has become one of the most common causes of death in patients with critical studies., more than 25% of the patients with sepsis complicated with cardiovascular complications, myocardial injury is an important indicator of prognosis of sepsis patients with sepsis. At present there is no adverse clinical targeted measures to alleviate the myocardial injury, so how to protect the cardiac function, to improve the physical condition of patients with severe sepsis, and increase the cure rate of.NF- has an important significance of nuclear factor kappa B (nuclear factor-kappa B) is a hot topic of current research, play a key role in the process of inflammation. Studies have confirmed that sepsis, NF- kappa B activity significantly increased, the application of siRNA (Small interfering Ribonucleic Acid) P65 NF- inhibitory kappa B subunit can effectively inhibit the inflammatory reaction. The traditional way to medicine siRNA is degradable in vivo. Nanotechnology can not only effectively protect the siRNA, can be obtained after chemical modification by active targeting function. _1- beta adrenergic receptor (beta _1-Adrenergic beta Receptors, _1-AR) in the human body are mainly distributed in the heart, myocardial injury, and the number of sensitive membrane receptors were significantly up-regulated, as the effect of targeting nanoparticles. Lipophilic compounds 3-{4-[2- hydroxy - (1- methyl ethylamine) propoxy] phenyl} sixteen ester propionic acid (PAC) and _1-AR receptor selective beta blocker esmolol (esmolol) has a similar chemical structure, previous studies showed that PAC liposomes modified with myocardial cell specific targeting to strong. So this research idea, using RNA interference technology and nano composite material Targeting P65-NF- kappa B-siRNA myocardial cells target beta _1-AR mediated in vitro to nanoparticles, and through intravenous injection into mice, observe the in vivo distribution, establish the model of acute myocardial injury of endotoxin, the myocardial protection, so as to alleviate sepsis myocardial injury in sepsis and protect the myocardial function, improve the cure rate of sepsis. Methods 1. esmolol PAC analogues synthesized by literature review and summarize and exchange, the 3- (4- hydroxyphenyl) propionic acid as the starting material, in turn through hydroxyl groups and sixteen alkyl alcohol ester, epoxy propyl, isopropyl amine etc. step, finally get the design and synthesis of the end product PAC.2.siRNA synthesis route and screening for P65 gene, designed and synthesized 3 kinds of siRNA and negative control siRNA, using lipo2000 siRNA were transfected into mouse myocardial cell line ( HL-1) cells, after 24h extraction of RNA PCR amplification, detected gene expression, we choose the best siRNA.3. nanoparticles synthesized by the literature and experience, to determine the siRNA+ of polyethylene glycol (polyethylene glycol, PEG) as the core, with PLGA [Poly (lactic-co-glycolic acid). PLGA] nano materials, with vitamin E polyethylene glycol succinate (Vitamin E-Tocopheryl Polyethylene Glycol 1000Succinate, TPGS) for the production method of emulsifier, ratio of reagents and optimization steps, preparation of myocardial targeted nanoparticles by laser particle size analyzer, Zeta potential analyzer, the characterization analysis of scanning electron microscope, measured and calculated nanoparticles by UV spectrophotometry and the entrapment efficiency, drug loading, release rate and a series of indicators. Through single factor analysis to optimize the preparation process, in order to get The in vitro properties of optimal nanoparticles.4. nanoparticles in cultured rat myocardial cells line (HL-1), the expression of fluorescence microscopy to detect cell surface receptor beta _1-AR. Each group of nanoparticles in the cell culture in suspension, the distribution and uptake of siRNA fluorescence microscopy and flow cytometry assay for nanoparticles in cells Si. NC and si435 nanoparticles were transfected into cells, cultured 24h after extraction of RNA was amplified by PCR, specific gene expression in animal experiments of.5. nanoparticles of different nanoparticles by tail vein injected into C57BL/6J mice, 24h mice after intraperitoneal injection of LPS (10mg/Kg) to establish sepsis injury model, small animal imaging detection the distribution of nanoparticles in vivo; apical puncture serum myocardial enzymes changes, inflammatory factors, cardiac tissues were obtained and tissue apoptosis gene detection Slice staining to identify myocardial injury. Results: 1. synthesis of esmolol analogue PAC high purity, stable property, biological toxicity, and can be used for the lowest expression level of RNA m further study on the synthesis of.2. transfected si435 cells, indicating that si435 of NF- kappa B subunit P65 inhibited by the strongest, the final choice of si435 as the core of myocardium targeting of.3. nanoparticles synthesized via analysis and comparison of different ratios of 875ul, 5%PEG+125ul siRNA 80mg PLGA+15mg TPGS+5mg as internal aqueous phase, PAC dissolved in 8ml acetone as the organic phase, 100ml 0.03%TPGS as the external phase, according to the particle according to the ratio of the synthesis of uniform size, good dispersion, potential distribution, the encapsulation rate of loading, the release curve is relatively stable myocardial cells of.4. mice (HL-1) cell surface expression of beta _1-AR receptor; co cultured cells and blank nanoparticles of different concentration, fine The cell survival rate was not affected, indicating no toxic nanoparticles on cells; distribution of fluorescence microscopy and flow cytometry were detected in cells, found nanoparticles evenly distributed, the uptake rate is high, stable inside the cell and have obvious inhibitory effect of P65.5. myocardial targeting nanoparticles have strong myocardial cell targeting and that nanoparticles can target recognition of myocardial cells; mice by intraperitoneal injection of LPS (10mg/Kg) can lead to myocardial injury obviously, and the degree of myocardial injury with si435 nanoparticles group were significantly reduced; sepsis, CK, LDH, HBDH were significantly increased, the expression of nanoparticles protection group decreased significantly; the expression of inflammatory cytokines. Apoptosis also showed the same trend, indicating that si435 nanoparticles can effectively protect myocardial cells, reducing myocardial injury in sepsis. The conclusion of the study 1. successful synthesis of esmolol is similar PAC, high purity, stable property, biological toxicity, and can be used for the synthesis of.2. further study we choose the best siRNA si435, expression of.3. could inhibit NF- kappa B subunit P65 synthesized PAC-P65-NF- kappa b-siRNA myocardial targeting nanoparticles, stable properties, uniform particle size, high encapsulation rate drug loading, good performance, stable release curve of.4. nanoparticles without biological toxicity to the cells, the siRNA package can release to myocardial cells and the interference effect, the damage degree of sepsis and inflammatory reaction of.5. nanoparticles can significantly improve the myocardial tissue, reduce inflammation, thereby protecting the heart function.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R459.7

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