DNA损伤修复蛋白NBS1在BPD新生大鼠模型中表达及其作用的研究

The expression and effect of DNA damage repair protein NBS1 in neonatal rats with bronchopulmonary dysplasia

目的研究DNA损伤修复蛋白奈梅亨断裂综合征蛋白1(Nijmegen breakage syndrome protein 1,NBS1)在新生大鼠支气管肺发育不良(bronchopulmonary dysplasia,BPD)模型中的动态表达规律,探讨其对BPD疾病发生、发展的影响。方法新生大鼠在生后12 h内随机分为BPD模型组50只和对照组50只,模型组吸入氧浓度为80%~85%,对照组吸入空气,两组分别于1 d、3 d、7 d、10 d、14 d收集肺组织标本,分离并培养肺泡Ⅱ型上皮细胞(alveolar typeⅡepithelial cell,AECⅡ)。光镜下观察肺组织形态学、辐射状肺泡计数(radial alveolar count,RAC)评价肺泡发育,免疫组织化学法及细胞免疫荧光观察NBS1的定位及表达,蛋白质免疫印迹、实时荧光定量PCR方法检测NBS1的表达水平。结果与对照组相比,模型组RAC值从生后7 d开始显著降低(对照组7.58±1.24,模型组5.42±1.24,P<0.01)。免疫组织化学法可见NBS1蛋白主要定位在肺泡上皮细胞的细胞核内,模型组1 d时主要在细胞核内表达,随暴露时间延长,胞质染色细胞数量增多,核内表达减弱。细胞免疫荧光可见NBS1蛋白在AECⅡ中主要定位于细胞核内,与对照组相比,模型组3 d开始细胞质染色增强,细胞核染色逐渐减弱,14 d时主要定位于细胞质。蛋白质免疫印迹结果显示,与对照组相比,模型组NBS1的蛋白表达于生后1d达高峰(对照组0.72±0.29,模型组1.28±0.22,P<0.01),随后逐渐下降,14 d表达低于对照组(对照组0.73±0.19,模型组0.49±0.11,P<0.05)。同样与对照组相比,模型组NBS1的mRNA表达水平于生后1 d达高峰(对照组1.00±0.00,模型组1.18±0.06,P<0.01),随后逐渐下降,14 d表达低于对照组(对照组1.07±0.13,模型组0.76±0.11,P<0.05)。结论在新生大鼠BPD模型中,NBS1蛋白的表达下调及核富集障碍可能影响了DNA损伤应答,这可能是介导BPD氧化应激损伤发生的机制之一。

Objective To investigate the dynamic expression of DNA damage repair protein Nijmegen breakage syndrome protein 1(NBS1)in the neonatal rats with bronchopulmonary dysplasia(BPD),and its influence on the development and progression of BPD.Methods Newborn rats were randomly divided into the BPD model group(n=50)and the control group(n=50)within 12 h after birth.The inhaled oxygen concentration was 80%-85%in the model group,and the control group inhaled air.In the two groups,lung tissue samples were collected on days 1,3,7,10 and 14,and isolated,purified and cultured alveolar epithelial typeⅡcells(AECⅡ).We observed pulmonary morphological changes under light microscope and evaluated alveolar development degree by radiate alveolar counts(RAC).Immunohistochemistry and cell immunofluorescence were used to observe the localization and expression of NBS1.Western blot and real-time quantitative PCR were used to detect the expression level of NBS1.Results Compared with the control group,the RAC value in the model group was decreased significantly from 7 d after birth(control group 7.58±1.24,model group 5.42±1.24,P<0.01).Immunohistochemistry showed that NBS1 protein was mainly located in the nucleus of alveolar epithelial cells.In the model group,NBS1 was mainly expressed in the nucleus on the 1st day.With the prolonged exposure time,the number of cytoplasmic staining cells increased and the expression in the nucleus decreased.Cell immunofluorescence farther showed that NBS1 protein was mainly located in the nucleus in AECⅡ.Compared with the control group,cytoplasmic staining in model group was enhanced from 3 d,while nuclear staining was gradually weakened,and was mainly located in the cytoplasm at 14 d.Western blot results showed that the expression of NBS1 protein in the model group peaked at 1 d compared to the control group(control group 0.72±0.29,model group 1.28±0.22,P<0.01),and then gradually decreased,with lower expression at 14 d compared to the control group(control group 0.73±0.19,model group 0.49±0.11,P<0.05).Similarly,the mRNA expression level of NBS1 in the model group peaked at 1 d compared to the control group(control group 1.00±0.00,model group 1.18±0.06,P<0.01),and then gradually decreased,with lower expression at 14 d than that in the control group(control group 1.07±0.13,model group 0.76±0.11,P<0.05).Conclusion In the neonatal rats with BPD,the down-regulation expression and nuclear enrichment disorder of NBS1 may affect the DNA damage response and be one of the mechanisms mediating the onset of oxidative stress damage in BPD.