本文選題：急性肺損傷 + 核因子κB��； 參考：《第四軍醫(yī)大學(xué)》2013年博士論文

【摘要】：背景：國內(nèi)外關(guān)于ALI/ARDS的研究大多數(shù)集中于探討細胞因子網(wǎng)絡(luò)的失衡，盡管使用各種藥物針對細胞因子進行了大量的臨床防治，，但始終沒有一種完全有效、可重復(fù)、有統(tǒng)計學(xué)意義的方法。ALI是一種炎癥免疫信號轉(zhuǎn)導(dǎo)紊亂引起的疾患。當(dāng)機體受到創(chuàng)傷、休克等損傷時，肺是首先被攻擊的靶器官和細菌來源之一。而肺泡巨噬細胞是肺組織炎癥瀑布效應(yīng)的激活點。核因子κB （NF-κB）是核轉(zhuǎn)錄調(diào)節(jié)蛋白的一種，具有多向調(diào)節(jié)的特點；參與多種炎性介質(zhì)基因和及多種免疫相關(guān)基因的轉(zhuǎn)錄和調(diào)控，是炎癥免疫反應(yīng)中多個信號途徑的匯聚點。本課題組前期研究證實NF-κB的核移位是誘導(dǎo)炎癥基因表達的關(guān)鍵環(huán)節(jié)，并在失控性炎癥反應(yīng)（SIRS）和急性肺損傷（ALI）的病理機制中發(fā)揮關(guān)鍵作用。但目前拮抗NF-κB活化通路、抑制炎癥損傷尚無特效藥物和成熟手段。 目的：為創(chuàng)傷性ALI篩選精確的基因藥物靶點，尋求一種高效、副作用小的炎癥靶向控制方法和尋求高效合理的治療方案提供實驗基礎(chǔ)和理論依據(jù)。 方法：本實驗運用已成功建立的家兔ALI模型，選定NF-κB信號通路的關(guān)鍵環(huán)節(jié)為突破口，利用載體/藥物技術(shù)，對體內(nèi)外炎癥效應(yīng)細胞（如肺泡巨噬細胞）的NF-κB核移位識別目的基因κB序列的 瓶頸‖通路進行特異性靶向封閉；并用分子生物學(xué)、免疫學(xué)和形態(tài)學(xué)等方法證實靶向封閉炎癥信號通路、調(diào)控炎癥基因表達防治SIRS/ALI的可行性和有效性。 結(jié)果：體外實驗：脂質(zhì)體轉(zhuǎn)染的雙鏈寡聚脫氧核苷酸（dsODNs）能有效降低肺泡巨噬細胞中炎癥介質(zhì)IL-1α、IL-6、IL-10、TNF-α和VCAM的mRNA表達和肺泡巨噬細胞培養(yǎng)液中LDH的含量（P0.01）；dsODNs-decoy/脂質(zhì)體為1:5時轉(zhuǎn)染效率最高，轉(zhuǎn)染6小時后細胞攝取率最高。為體內(nèi)試驗的順利進行奠定了基礎(chǔ)。 體內(nèi)試驗：雙鏈寡聚脫氧核苷酸（dsODNs-decoy）能有效減少肺組織的炎癥介質(zhì)IL-1α、IL-6、IL-10、TNF和VCAM的mRNA表達，dsODNs-decoy組與對照組和錯配dsODNs-decoy組比較有統(tǒng)計學(xué)意義；血清LDH檢測實驗組明顯減少（p0.01）；HE染色顯示對照組和錯配dsODNs-decoy組中肺泡變形、融合，大小不一；掃描電鏡顯示對照組和錯配dsODNs-decoy組肺泡隔增粗、增厚，肺泡隔不完整、斷裂。Western blot顯示dsODNs-decoy組中NF-κB p65核移位明顯減少。 結(jié)論：體外實驗中脂質(zhì)體轉(zhuǎn)染dsODNs-decoy具有較高的轉(zhuǎn)染效率，脂質(zhì)體轉(zhuǎn)染的dsODNs-decoy能有效降低對肺泡巨噬細胞的毒性。體內(nèi)試驗霧化吸入脂質(zhì)體轉(zhuǎn)染的dsODNs-decoy能有效減輕ALI的癥狀和炎性介質(zhì)的表達，并且血清LDH明顯降低，預(yù)示全身性副反應(yīng)小。
[Abstract]:Background: most of the studies on ALI/ARDS at home and abroad focus on the imbalance of cytokine network. Although a large number of clinical prevention and treatment of cytokines have been carried out with various drugs, none of them is completely effective and repeatable. Ali is a disorder caused by inflammatory immune signal transduction disorder. When the body is injured by trauma, shock, the lung is the first target organ and one of the source of bacteria. Alveolar macrophages are the activation point of inflammatory waterfall effect in lung tissue. Nuclear factor 魏 B (NF- 魏 B) is a kind of nuclear transcriptional regulatory protein, which is involved in the transcription and regulation of many inflammatory mediators and immune-related genes, and is the convergence point of many signaling pathways in inflammatory immune response. Our previous study confirmed that nuclear translocation of NF- 魏 B is a key link in inducing inflammatory gene expression and plays a key role in the pathological mechanism of uncontrolled inflammatory response (SIRS) and acute lung injury (ALI). However, there are no effective drugs and mature methods to antagonize NF- 魏 B activation pathway and inhibit inflammatory injury. Objective: to provide experimental and theoretical basis for screening accurate gene drug targets for traumatic ALI, seeking a highly effective and less side effect method for the control of inflammatory targets and seeking effective and reasonable therapeutic schemes. Methods: in this experiment, we selected the key link of NF- 魏 B signaling pathway as the breakthrough point and used carrier / drug technology to establish rabbit ALI model successfully. The NF- 魏 B nuclear translocation of inflammatory effector cells (such as alveolar macrophages) in vivo and in vitro was specifically targeted to block the bottleneck pathway of the target gene 魏 B sequence, and was used in molecular biology. Immunological and morphological methods confirmed the feasibility and effectiveness of blocking the inflammatory signaling pathway and regulating the expression of inflammatory genes in the prevention and treatment of SIRS/ALI. Results: in vitro experiment, the double-stranded oligodeoxynucleotide (dsODNs) transfected by liposome could effectively reduce the expression of mRNA and VCAM in the inflammatory mediators IL-1 偽, IL-10, TNF- 偽 and VCAM in alveolar macrophages, and the content of LDH in the culture medium of alveolar macrophages. The transfection efficiency was highest at 1:5. After transfection for 6 hours, the cell uptake rate was the highest. It lays a foundation for the in vivo test. In vivo test: dsODNs-decoy) could effectively reduce the expression of IL-1 偽, IL-6, IL-10, TNF and VCAM mRNA in lung tissue. Compared with the control group and mismatched dsODNs-decoy group, the expression of dsODNs-decoy was significantly lower in the DS-ODNs-decoy group than in the control and mismatched dsODNs-decoy groups. Serum LDH detection significantly reduced alveolar deformation, fusion and size in the control group and mismatched dsODNs-decoy group by HE staining, scanning electron microscopy showed that the alveolar septum was thickened and thickened, and the alveolar septum was incomplete in the control group and mismatched dsODNs-decoy group. Western blot showed that the nuclear translocation of NF- 魏 B p65 was significantly decreased in dsODNs-decoy group. Conclusion: liposome transfection of dsODNs-decoy has a high transfection efficiency in vitro. Liposome transfected dsODNs-decoy can effectively reduce the toxicity to alveolar macrophages. In vivo atomization inhalation of liposome-transfected dsODNs-decoy could effectively alleviate the symptoms of ALI and the expression of inflammatory mediators, and the serum LDH level was significantly decreased, indicating that the systemic side effects were small.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R563.8

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