阴离子交换转运蛋白Pendrin在急性肺损伤小鼠模型中的作用和干预研究

发布时间：2018-02-04 17:58

  本文关键词： Pendrin 脂多糖 急性肺损伤 肺泡Ⅱ型上皮 醋甲唑胺　出处：《北京协和医学院》2016年博士论文　论文类型：学位论文

【摘要】：背景：研究表明急性肺损伤/ARDS时患者存在气道尤其是小气道的功能受损和障碍,如气道阻力升高,产生存在内源性呼气末正压和/或呼吸系统压力-容积曲线拐点较低等,其与气道炎症、细胞外基质重塑和上皮剥脱有关。且研究证实气道上皮和肺泡上皮在炎症发生发展的演变过程中存在相似的生物学特性,急性肺损伤/ARDS时气道上皮的炎症等过程可能对ARDS的发生发展发挥重要作用。故气道上皮细胞可能可作为靶点用于发掘治疗急性肺损伤/ARDS的新药物和新的治疗策略。现已明确存在于气道上皮的阴离子转运蛋白Pendrin (SLC26A4)参与哮喘、COPD、百日咳等呼吸系统疾病的病生理学过程。至今,尚无Pendrin (SLC26A4)在急性肺损伤/ARDS作用和地位的研究。Pendrin (SLC26A4)是否在肺泡上皮表达也不得而知。本研究的目的旨在揭示Pendrin (SLC26A4)在急性肺损伤/ARDS的作用,同时明确该蛋白是否在肺泡上皮表达,明确气道上皮Pendrin (SLC26A4)是否可作为靶点用于急性肺损伤的治疗。方法：气管内滴注LPS诱导构建急性肺损伤C57BL/6小鼠模型。利用ELISA、CBA流式技术等检测肺组织和BALF中炎症指标,如组织学、炎症因子、BALF总蛋白和白细胞含量、肺水肿程度等评估模型。应用RT-PCR和Western蛋白印迹、免疫组化、免疫荧光技术检测分析Pendrin在肺组织中的转录和表达,并利用免疫荧光双标定位技术确定肺泡上皮是否存在Pendrin的表达。LPS造模后1h用Pendrin的抑制剂醋甲唑胺(5mg/kg)皮下注射,每12h重复一次。应用ELISA、CBA流式技术等评估分析肺部急性炎症指标。每组实验均设计对照,并重复3次。模型和干预的观察时长均为48h。应用GraphPad Prism 6.0软件进行数据统计分析和作图,符合正态分布的数据资料采用配对/非配对t检验分析比较组间差异,对不符合正态分布的数据进行变量转换为正态分布进行分析,P0.05认为具有统计学差异。结果：气管内滴注LPS可稳定构建急性肺损伤小鼠模型。与PBS组相比,LPS模型组肺组织SLC26A4基因的转录和Pendrin蛋白的表达明显上调。醋甲唑胺干预可减轻缓解肺部弥漫性肺泡损伤的病理改变,降低肺组织和BALF中炎症因子IL-6和MCP-1的浓度,减少肺组织MPO的分泌,使BALF中总蛋白含量和肺湿干比降低。醋甲唑胺干预后BALF中分类细胞计数巨噬细胞的比例增加。利用免疫荧光双标技术在ATⅡ细胞上定位到Pendrin的少量表达。结论：Pendrin可能参与LPS诱导的急性肺损伤的炎症病理过程。作为治疗靶点,抑制Pendrin的表达和功能可用于急性肺损伤的治疗。Pendrin在肺泡上皮的少量表达及其作用有待进一步揭示。该结果提示气道上皮细胞可作为靶点用于发掘治疗急性肺损伤/ARDS的新药物和新的治疗策略。
[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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