摘要
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�
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： （5
出处
《中华急诊医学杂志》
CAS
CSCD
2008年第11期1158-1162,共5页
Chinese Journal of Emergency Medicine
基金
家自然科学基金资助项目（30471824）
