γ泌肽酶抑制剂对胎鼠肺泡Ⅱ型上皮细胞Notch信号通路的影响

Effect of Gamma-Secretase Inhibitor on Notch Signaling Pathway in Alveolar Epithelial Type Ⅱ Cells of Preterm Rat

目的探讨γ泌肽酶抑制剂在肺泡Ⅱ型上皮细胞(AECⅡ)与肺成纤维细胞(LF)共培养胎鼠模型中对Notch信号分子表达的影响。方法应用Millipore插入式培养皿和Costar6孔板构建AECⅡ/LF共培养胎鼠模型。应用免疫组织化学法分别检测其细胞表面标志物表面活性蛋白C(SP-C)鉴定AECⅡ、LF。锥虫蓝拒染实验检测AECⅡ、LF活力后将γ泌肽酶抑制剂加入最低必需培养基(MEM)(终质量浓度为0.2 g/L)作为实验组,未加入者为对照组。应用反转录聚合酶链反应(RT-PCR)、蛋白质印迹法(Western blot)在基因和蛋白水平检测二组在培养24、48、72、96 h AECⅡNotch受体、转录调节因子DNA结合蛋白(CBF-1)及转录因子分裂增强子(HES-1)表达。采用SPSS11.5软件进行组间t检验和组内单因素方差分析。结果原代培养AECⅡ纯度(94.7±1.9)%,细胞活力为(96.2±2.5)%;LF纯度(97.2±1.4)%,细胞活力为(98.5±2.6)%。实验组CBF-1 mRNA及CBF-1蛋白在24、48、72、96 h与对照组比较均无显著变化(Pa>0.05),实验组Notch1 mRNA在24、48、96 h,Notch3 mRNA在48、72、96 h较对照组均显著降低(Pa<0.05),HES-1 mRNA及蛋白在各时间点均显著降低(Pa<0.05,0.01)。结论γ泌肽酶抑制剂可部分阻断胎鼠AECⅡ发育中的Notch信号通路传导,其机制可能通过非CBF-1依赖途径。

Objective To explore the inhitition effect of gamma - socretase inhibitor on Notch signaling molecules in alveolar epithelial type Ⅱ cell （ AECⅡ ） in a heterocellular culture of AEC Ⅱ and lung fibroblast（LF）. Methods By utilizing Millipore snap - on plate and Costar six - well plate, the AEC Ⅱ / LF co - culture system was established , After the AEC Ⅱ and LF were identified separately by surfactant protein C（ SP- C） and vimentin with immunohistochemical and evaluated with trypanblau assay, O. 2 g/L gamma -socretase inhibitor was added into minimal essential medium as experimental group and those without gamma - socretaso inhibitor as control group. Reverse transcription polymeraso chain reaction and Western blot were used to detect Notch1, Notch.3 receptors, transcription regulator C - promoter binding protein - 1 （CBF - 1 ） and clown stream transcription factor hairy/enhancer of split - 1 （HES - 1 ） at mRNA and protein level respectively at time poins 24,48,72,96 hours of culture. Results Primary cultured AEC Ⅱ showed a purity of （94.7 ± 1.9）% and uitality of （96.2 ±2.5 ） % ,while those of LF were （97.2± 1.4 ） % and （98.5 ± 2.6） %. CBF - 1 of experimental group showed no differences compared with that of control group（Pa 〉0.05）at 24,48,72,96 h, while Notchl mRNA of the experimental group decreased at 24,48,96 h and so did Notch3 mRNA at 48,72,96 h respectively compared with that of control group （ Pa 〈 0. 05 ）, HES - 1 mRNA and its protein decreased significantly at every time （Pa〈0. 05,0.01 ）. Conclusions Gamma - secretase inhibitor can partly inhibits Notch signaling pathway, which may go through CBF- 1 independent Notch signaling.