高氧对胎鼠肺泡Ⅱ型上皮细胞Notch受体的影响

Effect of hyperoxia on Notch receptor in alveolar epithelial type Ⅱ cells of preterm rats

目的检测Notch 1在肺泡Ⅱ型上皮细胞（ACEⅡ）与肺成纤维细胞共培养的高氧损伤模型中表达的变化，初步探讨Notch信号在高氧肺损伤中的作用，为临床防治新生儿急、慢性肺损伤提供理论的依据。方法SpragneDawkey雌鼠12只，体质量200，220g，雄鼠3只，220～250g，由华中科技大学同济医学院实验动物中心提供。将共培养的AECⅡ随机分为高氧组和对照组。高氧组按3L／min通入950ml／L O2和250ml／L CO2，10min后，立即用封口胶密封培养板，置于37℃、50ml／L CO2培养箱。于给氧后24、48、72、96h，收获各组细胞，测氧仪检测培养瓶中的O2体积分数，将低于900ml／LO2的标本弃去。免疫组织化学法鉴定细胞，台盼蓝拒染实验计算细胞纯度，确认共培养造模成功。每24h更换培养基并给氧。每一时间点同时设对照组，置于37℃、50ml／L CO2培养箱中培养。MTT法检测AEC Ⅱ增殖活力，绘制生长曲线；免疫组织化学法定位Notch1蛋白；荧光定量PCR检测notch1 mRNA；流式细胞术双标记法检测AECⅡ、AECⅠ细胞百分率。结果免疫组织化学法显示高氧组Notch1活化人核受抑；高氧组各时间点Notch1 mRNA分别下降为对照组的0．43、0．29、0．11、0．03倍（置信限95％），通氧后AECⅡ百分率显著降低[24h高氧组vs．对照组：（68．92±6．88）％vs．（90．35±4．01）％，P=0．006；48h高氧组vs．对照组：（38．03±3．27）％vs．（61．47±4．81）％，P=0．000；72h高氧组vs．对照组：（20．13±4．45）％vs．（52．05±3．35）％，P=0．000；96h高氧组vs．对照组：（8．17±1．99）％vs．（52．59±2．93）％，P：0．000]。AECⅠ百分率除96h外均增高[24h高氧组vs．财照组：（0．11±0．03）％vs．（0．01±0．01）％，P=0．006；48h高氧组vs．对照组：（49．73±3．45）％vs．（16．13±2．13）％，P=0．000；72h高氧组vs．对照组：（52．43±3．14）％vs．（5．98±0．95）％，P=0．000；96h高氧组vs．对照组：（19．85±3．26）％vs．（29．03±3．16）％，P=0．007]。结论高氧可抑制AECⅡNotch1受体的活化，引起AECⅡ增殖减弱，转分化异常。研究如何调控Notch信号通路将为修复肺泡上皮，恢复其正常生理功能打开新思路。

Objective To observe the effects of hyperoxia on Notch 1 receptor of alveolar epithelial type Ⅱ cells （AECⅡ ）, in a heterocellular culture of alveolar epithelial type Ⅱ cells and lung fibroblasts（LF）, in order to explore Notch signaling in hyperoxic induced lung injury and thus make theoretical basis for prevention and treatment of a acute / chronic neonatal lung injury. Method Twelve Spragne Dawkey female rats with 200 - 220 g and 3 Spragne Dawkey male rats with 220 - 250 g were offered from experimental animal centre of Tongji Medical Colleege, Huazhong University of Science and Technology. The AEC Ⅱ/ LF co-culture system was established successfully. AEC Ⅱ s from premature rats were randomly assigned to 2 groups： air control group and hyperoxia group. Air control group was kept in room air 50% ml/L CO2 environment at 37℃, while hyperoxia group was exposed to 950 ml/L O2 ＋ 250 ml/L CO2. Immuno-histochemistry was taken to detect Notch 1. Fluorescent quantitaive PCR was used to quantify the Notch 1 mRNA. MTT method was taken to assess cell proliferation viability. Flow cytometry double label method was used to detect cell percentages. Results In hyperoxia group： Notch 1 activation was inhibited, and Notch 1 mRNA decreased to 0.43,0.29,0.11,0.03 fold of control （95% confidence limit）. AEC Ⅱ percentage descended predominantly[24 h hyperoxia group vs. control group： （68.92 ± 6.88）% vs. （90.35± 4.01）%, P = 0.006;48 h hyperoxia group vs. control group： （38.03 ± 3.27） vs. （61.47 ± 4.81 ） %, P = 0.000;72 h hyperoxia group vs. control group： （20.13 ± 4.45） % vs. （52.05 ± 3.35） %, P = 0.000;96 h hyperoxia group vs. control group：（8.17± 1.99）% vs. （52.59±2.93）%, P = 0.0003 while that of AEC Ⅰ rised[24 h hyperoxia group vs. control group： （0.11 ± 0.03） % vs. （0.01 ±0.01） %, P = 0.006;48 h hypemxia grout） vs. control group： （49.73 ± 3.45） % vs. （ 16.13 ± 2.13） %, P = 0.000;72 h hyperoxia group vs. control group： （52.43 ± 3.14） % vs. （5.98 ± 0.95） %, P = 0.000;96 h hyperoxia group vs. control group： （19.85 ± 3.26）% vs. （29.03 ± 3.16）%, P = 0.007]. Conclusions Hyperoxia may iuhibit Notch signaling pathway, which can weaken proliferation and disdifferentiation of AEC II s. Investigations on how to control Notch signaling will provide fresh thoughts for alveolar epithelium repairing.