不同氧浓度暴露对早产鼠肺泡Ⅱ型上皮细胞的损伤及CGRP的保护效应

Protection of Calcitonin Gene-Related Peptide From Damage Induced by Hyperoxia on Premature Rat Type Ⅱ Alveolar Epithelial Cells

目的观察不同氧浓度对早产鼠肺泡Ⅱ型上皮细胞(AECⅡ)的影响以及降钙素基因相关肽(CGRP)对AECⅡ的保护作用。方法将原代分离培养的孕19天早产鼠AECⅡ接种至6孔培养板,随机分为空气组、40%氧组、60%氧组、80%氧组及相关浓度氧CGRP组与相关浓度氧CGRP受体拮抗剂组。空气组和高氧组分别置于体积分数为21%的空气或40%、60%、80%的氧气中暴露24 h;CGRP组在暴露前加入CGRP;CGRP拮抗剂组在CGRP组基础上加入CGRP受体拮抗剂(CGRP8-37)。培养24 h后,用分光光度计测定各组丙二醛(MDA)、总抗氧化能力(TAOC)、超氧化物歧化酶(SOD)水平;用流式细胞仪检测活性氧(ROS)和细胞凋亡率;用逆转录—聚合酶链反应(RT-PCR)测定表面活性蛋白C(SP-C)的mRNA表达。结果与空气组比较,40%、60%、80%氧组MDA、ROS及细胞调亡率均显著增高,TAOC、SOD水平及SP-C mRNA表达均显著降低(P均<0.01)。与氧气组比较,高氧CGRP组细胞MDA、ROS水平及细胞调亡率均显著下降;而TAOC、SOD水平及SP-C mRNA表达均明显增高(P<0.01)。高氧CGRP拮抗剂组与高氧组各指标比较差异均无显著性。结论高于40%氧暴露24 h可导致早产鼠AECⅡ发生氧化损伤,诱导细胞凋亡及SP-C mRNA表达下降;随着氧浓度增加,损伤加重;而CGRP可部分减轻AECⅡ的氧化损伤,减少凋亡,促进SP-C mRNA表达,对高氧损伤的AECⅡ起保护作用。

Objective To explore the influence of different oxygen concentrations and the effects of calcitonin gene-related peptide（CGRP） on type Ⅱ alveolar epithelial cells（AECⅡ） isolated from premature rat lung in vitro.Methods AECⅡ were isolated from 19d fetal rat lung and cultured for 12h to attach.Then AECⅡ were randomly divided into ten groups：air group,40% oxygen concentration group,60% oxygen concentration group,80% oxygen concentration group,hyperoxia（40%,60%,80%） plus CGRP group,hyperoxia（40%,60%,80%）plus CGRP and CGRP8-37 group.AECⅡ of air group and hyperoxia group were exposed to 21%,40%,60%,80% oxygen respectively for 24h while hyperoxia plus CGRP group were added with CGRP and hyperoxia plus CGRP and CGRP8-37 group with CGRP and CGRP8-37（CGRP receptor antagonist） before placed into 40%,60%,80% oxygen concentration.Concentrations of maleic dialdehyde（MDA）,superoxide dismutase（SOD） and total antioxidant capacity（TAOC） in culture cells were detected by ultraviolet spectrophotometer.Reactive oxygen species（ROS） and apoptosis rate of AECⅡ were analyzed by flow cytometry and the mRNA level of surfactant associated protein C（SP-C） was measured by RT-PCR.Results The levels of MDA,ROS and apoptosis cell number were increased whereas TAOC,SOD and SP-C mRNA expression declined in hyperoxia group compared with those in air group（P0.01）.Reversely,MDA,ROS and apoptosis rate were significantly lower and levels of TAOC,SOD and SP-C mRNA expression were significantly higher in group hyperoxia plus CGRP group than in hyperoxia group or hyperoxia plus CGRP and CGRP8-37 group.Conclusions Exposure to 40%,60%,80% oxygen for 24h could cause oxidative injury,induce apoptosis and decrease SP-C mRNA level of AECⅡ in vitro in premature rats,while CGRP may play a protective role against hyperoxic lung injury by antioxidant,inhibition of AEC apoptosis and promotion of the SP-C mRNA expression.