本文關(guān)鍵詞： Pendrin 脂多糖 急性肺損傷 肺泡Ⅱ型上皮 醋甲唑胺　出處：《北京協(xié)和醫(yī)學(xué)院》2016年博士論文　論文類型：學(xué)位論文

【摘要】：背景：研究表明急性肺損傷/ARDS時(shí)患者存在氣道尤其是小氣道的功能受損和障礙,如氣道阻力升高,產(chǎn)生存在內(nèi)源性呼氣末正壓和/或呼吸系統(tǒng)壓力-容積曲線拐點(diǎn)較低等,其與氣道炎癥、細(xì)胞外基質(zhì)重塑和上皮剝脫有關(guān)。且研究證實(shí)氣道上皮和肺泡上皮在炎癥發(fā)生發(fā)展的演變過程中存在相似的生物學(xué)特性,急性肺損傷/ARDS時(shí)氣道上皮的炎癥等過程可能對(duì)ARDS的發(fā)生發(fā)展發(fā)揮重要作用。故氣道上皮細(xì)胞可能可作為靶點(diǎn)用于發(fā)掘治療急性肺損傷/ARDS的新藥物和新的治療策略�，F(xiàn)已明確存在于氣道上皮的陰離子轉(zhuǎn)運(yùn)蛋白Pendrin (SLC26A4)參與哮喘、COPD、百日咳等呼吸系統(tǒng)疾病的病生理學(xué)過程。至今,尚無Pendrin (SLC26A4)在急性肺損傷/ARDS作用和地位的研究。Pendrin (SLC26A4)是否在肺泡上皮表達(dá)也不得而知。本研究的目的旨在揭示Pendrin (SLC26A4)在急性肺損傷/ARDS的作用,同時(shí)明確該蛋白是否在肺泡上皮表達(dá),明確氣道上皮Pendrin (SLC26A4)是否可作為靶點(diǎn)用于急性肺損傷的治療。方法：氣管內(nèi)滴注LPS誘導(dǎo)構(gòu)建急性肺損傷C57BL/6小鼠模型。利用ELISA、CBA流式技術(shù)等檢測(cè)肺組織和BALF中炎癥指標(biāo),如組織學(xué)、炎癥因子、BALF總蛋白和白細(xì)胞含量、肺水腫程度等評(píng)估模型。應(yīng)用RT-PCR和Western蛋白印跡、免疫組化、免疫熒光技術(shù)檢測(cè)分析Pendrin在肺組織中的轉(zhuǎn)錄和表達(dá),并利用免疫熒光雙標(biāo)定位技術(shù)確定肺泡上皮是否存在Pendrin的表達(dá)。LPS造模后1h用Pendrin的抑制劑醋甲唑胺(5mg/kg)皮下注射,每12h重復(fù)一次。應(yīng)用ELISA、CBA流式技術(shù)等評(píng)估分析肺部急性炎癥指標(biāo)。每組實(shí)驗(yàn)均設(shè)計(jì)對(duì)照,并重復(fù)3次。模型和干預(yù)的觀察時(shí)長均為48h。應(yīng)用GraphPad Prism 6.0軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)分析和作圖,符合正態(tài)分布的數(shù)據(jù)資料采用配對(duì)/非配對(duì)t檢驗(yàn)分析比較組間差異,對(duì)不符合正態(tài)分布的數(shù)據(jù)進(jìn)行變量轉(zhuǎn)換為正態(tài)分布進(jìn)行分析,P0.05認(rèn)為具有統(tǒng)計(jì)學(xué)差異。結(jié)果：氣管內(nèi)滴注LPS可穩(wěn)定構(gòu)建急性肺損傷小鼠模型。與PBS組相比,LPS模型組肺組織SLC26A4基因的轉(zhuǎn)錄和Pendrin蛋白的表達(dá)明顯上調(diào)。醋甲唑胺干預(yù)可減輕緩解肺部彌漫性肺泡損傷的病理改變,降低肺組織和BALF中炎癥因子IL-6和MCP-1的濃度,減少肺組織MPO的分泌,使BALF中總蛋白含量和肺濕干比降低。醋甲唑胺干預(yù)后BALF中分類細(xì)胞計(jì)數(shù)巨噬細(xì)胞的比例增加。利用免疫熒光雙標(biāo)技術(shù)在ATⅡ細(xì)胞上定位到Pendrin的少量表達(dá)。結(jié)論：Pendrin可能參與LPS誘導(dǎo)的急性肺損傷的炎癥病理過程。作為治療靶點(diǎn),抑制Pendrin的表達(dá)和功能可用于急性肺損傷的治療。Pendrin在肺泡上皮的少量表達(dá)及其作用有待進(jìn)一步揭示。該結(jié)果提示氣道上皮細(xì)胞可作為靶點(diǎn)用于發(fā)掘治療急性肺損傷/ARDS的新藥物和新的治療策略。
[Abstract]:Background: studies have shown that patients with acute lung injury / ARDS have impaired airway function, especially small airway dysfunction, such as increased airway resistance. There were endogenous positive end-expiratory pressure and / or respiratory pressure-volume curve inflexion and so on, which were associated with airway inflammation. Extracellular matrix remodeling is related to epithelial exfoliation. It is confirmed that airway epithelium and alveolar epithelium have similar biological characteristics in the process of inflammation. Inflammation of airway epithelium during acute lung injury / ARDS may play an important role in the occurrence and development of ARDS, so airway epithelial cells may be used as targets to explore and treat acute lung injury / ARDS. New drugs and new therapeutic strategies. Pendrin, an anionic transporter present in airway epithelium. (. SLC26A4) was involved in asthma. Copd, whooping cough, and other respiratory diseases in the process of disease physiology. Up to now. There is no study on the role and role of Pendrin / SLC26A4 in acute lung injury / / ARDS.Pendrin / SLC26A4). The purpose of this study was to investigate the role of Pendrin in acute lung injury. At the same time, it was determined whether the protein was expressed in alveolar epithelium. Identification of airway epithelial Pendrin's SLC26A4). Methods: C57BL / 6 mouse model of acute lung injury was induced by intratracheal instillation of LPS. ELISA was used. CBA flow cytometry was used to detect the content of total protein and white blood cell in lung tissue and BALF. RT-PCR and Western Western blot, immunohistochemistry and immunofluorescence technique were used to detect the transcription and expression of Pendrin in lung tissue. Immunofluorescence double labeling technique was used to determine whether there was Pendrin expression in alveolar epithelium. 1 h after modeling, 5 mg / kg of Pendrin was used as an inhibitor of Pendrin. Subcutaneous injection. Acute pulmonary inflammation was evaluated by ELISA-CBA flow cytometry every 12 hours. The observation time of the model and intervention was 48 h. The data were statistically analyzed and mapped by GraphPad Prism 6.0 software. The data in accordance with normal distribution were analyzed by paired / unpaired t test, and the data that did not conform to normal distribution were transformed into normal distribution. Results: intratracheal instillation of LPS could stably construct acute lung injury model in mice. Compared with PBS group. In LPS model group, the transcription of SLC26A4 gene and the expression of Pendrin protein were upregulated obviously. The concentrations of inflammatory cytokines IL-6 and MCP-1 in lung tissue and BALF were decreased, and the secretion of MPO in lung tissue was decreased. The total protein content and lung wet / dry ratio of BALF were decreased. The percentage of macrophage count in BALF was increased after the intervention of acetazolamide. Pen was located on AT 鈪，

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