神经肽P物质调控Notch信号通路对胎鼠高氧肺损伤后Ⅱ型肺泡上皮细胞增殖的影响  

Effect of neuropeptide substance P regulates Notch signal pathway on the proliferation of the type Ⅱ alveolar epithelial cells in primary premature rats with hyperxia-induced lung injury

作　　者：蔡莉[1] 方芳[1] 杨林[1] 党红星[1] 许峰[1] 

机构地区：[1]重庆医科大学附属儿童医院PICU,儿童发育疾病研究省部共建教育部重点实验室,儿童发育重大疾病国家国际科技合作基地,儿科学重庆重点实验室400014

出　　处：《中华危重病急救医学》2016年第6期508-512,共5页Chinese Critical Care Medicine

基　　金：国家自然科学基金（81341021）

摘　　要：【摘要】目的探讨神经肽P物质（SP）对胎鼠高氧肺损伤后Ⅱ型肺泡上皮细胞（AECI／）增殖的影响及其可能机制。方法取孕18d胎鼠肺组织进行原代AEC11分离培养后分为6组。空气组暴露于含21％的氧舱内；高氧组则暴露于含95％的氧舱内；高氧＋SP组、高氧＋SP＋SP受体拮抗剂L703．606组、高氧＋SP＋Notch信号通路阻断剂MW167组、高氧＋MWl67组在高氧暴露基础上相应加入1μmol／L的SP、0．03mmol／L的L703．606、0．2mmol／L的MW167。干预后4h检测细胞活性，于6、12、18、24h计数细胞并描绘增殖曲线，24h于倒置显微镜下观察细胞形态及增殖情况并检测细胞周期；用实时荧光定量反转录-聚合酶链反应（RT—qPCR）检测Notch信号通路配体Dll1、D114以及靶基因Hes1的mRNA表达。结果经高氧或Notch信号通路阻断剂MW167处理后，各组细胞计数均较空气组明显减少，且细胞活性明显降低，出现G1期阻滞现象，细胞增殖能力明显受抑；镜下可见核固缩、胞内颗粒物减少等变化；同时Notch通路相关基因Dll1、D114、Hes1的mRNA表达量明显下降。与高氧组比较，SP干预后细胞计数增多，胞内颗粒物质增加，且细胞活性明显升高（A值：75．5±14．9比44．9±8.4），G1期比例降低[（62．87±3．99）％比（67．47±2．28）％]、S期比例升高[（32．54±2．71）％比（25．03±3．52）％]，说明细胞增殖能力明显增强（均P〈0．05）；同时Dll1、D114、Hes1的mRNA表达量均明显上调（2^-△△Ct：1．44±0．13比0．92±0．04，0．80±0．17比0．35±0．08，1．20±0．09比0．85±0．08，均P〈0．05）。而此时再加入SP受体拮抗剂L703．606或Notch信号通路阻断剂MW167，可再次出现细胞增殖受抑的表现，且细胞活性再次降低（A值：60．0±9．5、37．7±21．7比75．5±14．9），G1期比例增加[（73．96±3．53）％、（92．44±1．75）％比（62．87±3．99）�Objective To investigate the relationship between neuropeptide substance P （SP） on the regulatory of the proliferation of the type Ⅱ alveolar epithelial cells （AEC Ⅱ） in primary premature rats with hyperxia-induced lung injury and its possible mechanism. Methods The AEC Ⅱ of primary premature rats which pregnant 18 days were isolated and purified. The AEC Ⅱ were divided into six groups. The cells in air group were exposed in a closed oxygen cabin containing 21% oxygen, and those in hyperoxia group were exposed in 95% oxygen. The cells in hyperoxia ± SP group, hyperoxia ± SP ± SP antagonist L703.606 group, hyperoxia ± SP ± blocker of Notch signal pathway MW167 group and hyperoxia ± MW167 group were treated with 1 μmol/L of SP, 0.03 mmol/L of L703.606 or 0.2 mmol/L of MW167 on the basis of hyperoxia exposure. Cell activity was determined after intervention for 4 hours. The cells were counted at 6, 12, 18, and 24 hours, and growth curve was drew. Morphologic change and proliferation were observed under inverted microscope at 24 hours, and the cell cycle was determined with flow cytometry. The mRNA expressions of the ligands of Notch signal pathway Dill, Dl14 and its target gene Hesl were determined by real-time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Results The cell number with the intervened of hyperoxia or blocker of Notch signal pathway MWI67 was decreased significantly as compared with air group, the cell activity was decreased significantly, G1 phase arrest and obviously suppression of cell proliferation were appeared, and karyopyknosis and intracellular particulate matter less were observed with inverted microscope. The mRNA expressions of Dill, Dl14 and Hesl were decreased significantly. While the cell number was increased remarkably, the intracellular matter was increased, and the cell activity was obviously increased after SP intervention （A value： 75.5 ± 14.9 vs. 44.9 ± 8.4）, the proportion of G1 phase was reduced [（62.87 ±

关 键 词：神经肽P物质 NOTCH信号通路 肺泡上皮细胞 Ⅱ型 肺损伤 

分 类 号：R722.1[医药卫生—儿科]