本文關(guān)鍵詞： 膿毒癥 心肌損傷 納米粒 心肌靶向　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景膿毒癥(sepsis)是由細(xì)菌、真菌、病毒等感染因素導(dǎo)致的全身性炎癥反應(yīng),起病急驟、病情危重、機(jī)制復(fù)雜、預(yù)后較差是其重要特征,通常會引起全身多臟器功能障礙、衰竭,已成為危重患者的常見死亡原因之一。研究表明,25%以上的膿毒癥患者合并心血管并發(fā)癥,心肌損傷是膿毒癥患者預(yù)后不良的重要標(biāo)志。目前臨床上并無針對性的措施來減輕心肌損傷,因此如何保護(hù)心功能,對改善危重患者身體狀況、提高膿毒癥的救治率具有重要意義。NF-κB(nuclear factor-kappa B)是當(dāng)代研究的熱點(diǎn),在過度炎癥反應(yīng)發(fā)生過程中起關(guān)鍵性作用。研究證實(shí),膿毒癥發(fā)生時(shí),NF-κB的活性明顯升高,此時(shí)應(yīng)用siRNA(Small interfering Ribonucleic Acid)抑制NF-κB的P65亞基可有效抑制炎癥反應(yīng)。傳統(tǒng)的給藥方式中siRNA在體內(nèi)易被降解,納米技術(shù)不僅能有效保護(hù)siRNA,也能通過化學(xué)修飾后獲得主動靶向功能。β_1-腎上腺素受體(β_1-Adrenergic Receptors,β_1-AR)在人體內(nèi)主要分布于心臟,心肌損傷時(shí),膜表面受體的數(shù)量與敏感性均顯著上調(diào),可作為納米粒的效應(yīng)靶點(diǎn)。親脂性化合物3-{4-[2-羥基-(1-甲基乙胺基)丙氧基]苯基}丙酸十六醇酯(PAC)與β_1-AR受體選擇性阻滯劑艾司洛爾(esmolol)具有類似的化學(xué)結(jié)構(gòu),以往的研究表明PAC修飾的脂質(zhì)體具有較強(qiáng)的心肌細(xì)胞特異靶向性。故本課題設(shè)想,利用RNA干擾技術(shù)以及復(fù)合納米材料的靶向性,體外構(gòu)建β_1-AR介導(dǎo)的P65-NF-κB-siRNA心肌細(xì)胞靶向納米粒,并通過尾靜脈注射導(dǎo)入小鼠體內(nèi),觀察其在小鼠體內(nèi)的分布情況,建立急性內(nèi)毒素心肌損傷模型,研究其對心肌細(xì)胞的保護(hù)作用,從而實(shí)現(xiàn)減輕膿毒癥心肌損傷、保護(hù)心肌功能、提高膿毒癥救治率的目的。研究方法1.艾司洛爾類似物PAC的合成通過查閱文獻(xiàn)及總結(jié)交流,確定了以3-(4-羥基苯基)丙酸為起始原料,依次經(jīng)過羥基與十六烷醇成酯、環(huán)氧丙基化、異丙胺化等步驟,最后得到終產(chǎn)物PAC的合成路線。2.siRNA的設(shè)計(jì)合成與篩選針對P65基因,設(shè)計(jì)合成3種不同的siRNA及陰性對照siRNA,利用lipo2000將siRNA分別轉(zhuǎn)染到小鼠心肌細(xì)胞系(HL-1)細(xì)胞內(nèi),培養(yǎng)24h后提取RNA行PCR擴(kuò)增,檢測各組基因表達(dá)情況,篩選出干擾效果最好的siRNA。3.納米粒的合成通過查閱文獻(xiàn)和借鑒經(jīng)驗(yàn),確定了以siRNA+聚乙二醇(polyethylene glycol,PEG)為內(nèi)核、以聚乳酸-羥基乙酸共聚物[Poly(lactic-co-glycolic acid),PLGA]為納米材料、以維生素E聚乙二醇琥珀酸酯(Vitamin E-Tocopheryl Polyethylene Glycol 1000Succinate,TPGS)為乳化劑的制作方法,優(yōu)化試劑配比及操作步驟,制備心肌靶向納米粒,并利用激光粒徑儀、Zeta電位分析儀、掃描電子顯微鏡等進(jìn)行各表征分析,通過紫外分光光度計(jì)檢測并計(jì)算納米粒的包封率、載藥量、釋放率等一系列指標(biāo)。通過單因素分析優(yōu)化制備工藝,以得到性質(zhì)最佳的納米粒。4.納米粒的細(xì)胞實(shí)驗(yàn)體外培養(yǎng)大鼠心肌細(xì)胞系(HL-1),熒光顯微鏡檢測細(xì)胞表面β_1-AR受體表達(dá)情況。將各組納米粒加入細(xì)胞培養(yǎng)混懸液中,通過熒光顯微鏡和流式細(xì)胞學(xué)技術(shù)檢測細(xì)胞對納米粒的攝取及siRNA在細(xì)胞內(nèi)的分布情況。將si NC納米粒和si435納米粒分別轉(zhuǎn)染細(xì)胞,培養(yǎng)24h后提取RNA進(jìn)行PCR擴(kuò)增,明確基因表達(dá)情況。5.納米粒的動物實(shí)驗(yàn)將不同納米粒通過尾靜脈注射到C57BL/6J小鼠體內(nèi),24h后小鼠腹腔注射LPS(10mg/Kg)制作膿毒癥損傷模型,小動物成像儀檢測納米粒在體內(nèi)的分布情況;心尖穿刺取血檢測心肌酶譜、炎癥因子等的變化,取心臟組織行凋亡基因檢測和組織切片染色以明確心肌損傷情況。研究結(jié)果1.合成的艾司洛爾類似物PAC純度高,性質(zhì)穩(wěn)定,無生物學(xué)毒性,可用于進(jìn)一步的合成研究。2.轉(zhuǎn)染si435的細(xì)胞中m RNA水平表達(dá)最低,說明si435對NF-κB的P65亞基抑制作用最強(qiáng),最終選擇si435作為心肌靶向納米粒的核心。3.經(jīng)過不同配比的合成分析比對,875ul 5%PEG+125ul siRNA作為內(nèi)水相,80mg PLGA+15mg TPGS+5mg PAC溶于8ml丙酮作為有機(jī)相,100ml 0.03%TPGS作為外水相,按照此配比合成的納米粒粒徑均一,分散性好,電勢分布集中,此時(shí)的包封率,載藥量,釋放曲線也比較穩(wěn)定。4.小鼠心肌細(xì)胞系(HL-1)細(xì)胞膜表面有明顯β_1-AR受體表達(dá);不同濃度的空白納米粒與細(xì)胞共培養(yǎng),細(xì)胞存活率未受影響,說明納米粒對細(xì)胞無毒性;熒光顯微鏡和流式細(xì)胞學(xué)檢測納米粒在細(xì)胞內(nèi)的分布情況,發(fā)現(xiàn)納米粒分布均勻,細(xì)胞攝取率高,能穩(wěn)定進(jìn)入細(xì)胞內(nèi)部并產(chǎn)生明顯的P65抑制效應(yīng)。5.心肌靶向納米粒均有較強(qiáng)的心肌細(xì)胞靶向性,說明納米粒能靶向識別心肌細(xì)胞;小鼠腹腔注射LPS(10mg/Kg)可導(dǎo)致明顯的心肌損傷,而注射si435納米粒組的心肌損傷程度明顯減輕;膿毒癥發(fā)生時(shí),CK、LDH、HBDH均明顯升高,納米粒保護(hù)組的表達(dá)則顯著降低;炎癥因子、凋亡相關(guān)基因的表達(dá)情況也均有同樣的趨勢,說明si435納米粒能有效保護(hù)心肌細(xì)胞、降低膿毒癥心肌損傷。研究結(jié)論1.成功合成艾司洛爾類似物PAC,純度高,性質(zhì)穩(wěn)定,無生物學(xué)毒性,可用于進(jìn)一步的合成研究。2.篩選出干擾效果最好的siRNA—si435,能明顯抑制NF-κB P65亞基的表達(dá)。3.成功合成了PAC-P65-NF-Κb-siRNA心肌靶向納米粒,性質(zhì)穩(wěn)定,粒徑均一,包封率高,載藥性能好,釋放曲線穩(wěn)定。4.納米粒對細(xì)胞無生物學(xué)毒性,其包裹的siRNA能釋放到心肌細(xì)胞內(nèi)并產(chǎn)生干擾效應(yīng),減輕炎癥反應(yīng)。5.納米粒能顯著改善心肌組織的膿毒癥損傷程度,減輕炎癥反應(yīng),從而保護(hù)心臟功能。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 張振飛;王希柱;;膿毒癥心肌損傷機(jī)制研究及治療進(jìn)展[J];內(nèi)蒙古中醫(yī)藥;2013年22期

2 李美平;孫國杰;尤行宏;;頭針對心肌缺血再灌注大鼠心交感放電、心肌β_1-腎上腺素受體蛋白表達(dá)及血漿去甲腎上腺素含量的影響[J];針刺研究;2012年05期

3 陳妍,鄧英杰,郝艷麗,王振遠(yuǎn),盛軍;心肌細(xì)胞靶向脂質(zhì)體的制備及體外靶向性研究[J];藥學(xué)學(xué)報(bào);2005年06期

4 陳妍,鄧英杰,郝艷麗,王秀敏,王振遠(yuǎn),盛軍;膜修飾脂質(zhì)體的制備及對心肌細(xì)胞的靶向作用[J];沈陽藥科大學(xué)學(xué)報(bào);2005年02期

，

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