不同程度间歇低氧对小鼠肺成纤维细胞活化和胞外基质分泌的影响
|
摘要
目的探讨不同程度间歇低氧(IH)对MLg小鼠肺成纤维细胞活化和胞外基质分泌的影响。方法取对数生长期的MLg成纤维细胞,IH分别处理1 h、4 h、8 h(IH1组、IH4组、IH8组),IH频率为5%O_2100 s,21%O_2120 s,并设置常氧对照组(21%O_2处理8 h,N组)。实验结束后收集细胞,采用q RT-PCR和Western blot检测各组α肌动蛋白(α-SMA)、Ⅰ型胶质蛋白(COL1)m RNA和蛋白表达水平。结果 IH1组、IH4组、IH8组α-SMA、COL1 m RNA及蛋白的表达水平明显高于N组(P<0.05),且随着IH时间的延长,α-SMA、COL1表达水平逐渐升高。结论 IH可以促进小鼠肺成纤维细胞活化和胞外基质分泌,其作用机制可能与氧化应激有关。
Objective To explore the effects of different degrees of intermittent hypoxia(IH) on the activation and thesecretion of extracellular matrix in MLg lung fibroblast cell line. Methods MLg lung fibroblast cells in logarithmic growthphase were exposed for 5% O_2 for 100 seconds and 21% O_2 for 120 seconds in 1 h, 4 h and 8 h groups(IH1, IH4 and IH8)and normoxia group(21% O_2 for 8 h, N group). The cells in each group were collected at the end of experiment. Real-timePCR was used to measure the m RNA expression levels of α-SMA and type Ⅰ collagen(COL1) A1, and Western blot assaywas used to detect the protein expression levels of α-SMA and COL1. Results The m RNA and protein expression levels ofα- SMA and COL1 were significantly increased in IH1, IH4 and IH8 groups than those in N group(all P < 0.05).Furthermore, expression levels of α- SMA and COL1 showed a time- dependent increase with IH exposure time.Conclusion The intermittent hypoxia can promote the cell activation and the extracellular matrix secretion of mouse lungfibroblast cells, which may be related with the oxidative stress.
Objective To explore the effects of different degrees of intermittent hypoxia(IH) on the activation and thesecretion of extracellular matrix in MLg lung fibroblast cell line. Methods MLg lung fibroblast cells in logarithmic growthphase were exposed for 5% O_2 for 100 seconds and 21% O_2 for 120 seconds in 1 h, 4 h and 8 h groups(IH1, IH4 and IH8)and normoxia group(21% O_2 for 8 h, N group). The cells in each group were collected at the end of experiment. Real-timePCR was used to measure the m RNA expression levels of α-SMA and type Ⅰ collagen(COL1) A1, and Western blot assaywas used to detect the protein expression levels of α-SMA and COL1. Results The m RNA and protein expression levels ofα- SMA and COL1 were significantly increased in IH1, IH4 and IH8 groups than those in N group(all P < 0.05).Furthermore, expression levels of α- SMA and COL1 showed a time- dependent increase with IH exposure time.Conclusion The intermittent hypoxia can promote the cell activation and the extracellular matrix secretion of mouse lungfibroblast cells, which may be related with the oxidative stress.
引文
[1]Alves ES,Lira FS,Santos RV,et al.Obesity,diabetes and OSASinduce of sleep disorders:exercise as therapy[J].Lipids HealthDis,2011,10:148.doi:10.1186/1476-511X-10-148.
[2]黄绍光,李庆云.睡眠呼吸暂停低通气综合征的流行病学研究现状[J].中华全科医师杂志,2005,4(4):197-198.Huang SG,Li QY.Current status in epidemiological studies on sleep apnea-hypopnea syndrome[J].Chinese Joural of General Practitioners,2005,4(4):197-198.doi:10.3760/cma.j.issn.1671-7368.2005.04.001.
[3]Shamsuzzaman AS,Gersh BJ,Somers VK.Structive sleep apnea:implications for cardiac and vascular disease[J].JAMA,2003,290(14):1906-1914.doi:10.1001/jama.290.14.1906.
[4]Passali D,Corallo G,Yaremchuk S,et al.Oxidative stress inpatients with obstructive sleep apnoea syndrome[J].ActaOtorhinolaryngol Ital,2015,35(6):420-425.doi:10.14639/0392-100X-895.
[5]O’Dwyer DN,Ashley SL,Moore BB.Influences of innate immunity,autophagy,and fibroblast activation in the pathogenesis of lungfibrosis[J].Am J Physiol Lung Cell Mol Physio,2016,311(3):L590-601.doi:10.1152/ajplung.00221.2016.
[6]King TE Jr,Albera C,Bradford WZ,et al.Effect of interferongamma-1b on survival in patients with idiopathic pulmonaryfibrosis(INSPIRE):a multicentre,randomised,placebo-controlledtrial[J].Lancet,2009,374(9685):222-228.doi:10.1016/S0140-6736(09)60551-1.
[7]O’Halloran KD.Chronic intermittent hypoxia creates the perfectstorm with calamitous consequences for respiratory control[J].Respir Physiol Neurobiol,2016,226:63-67.doi:10.1016/j.resp.2015.10.013.
[8]韩苗苗,何庆,施遥,等.睡眠呼吸暂停模式间歇低氧对心脑血管的影响[J].天津医药,2014,42(9):946-949.Han MM,He Q,Shi Y,et al.The impact of intermittent hypoxia from obstructivesleep apnea on cardiovascular and cerebrovascular diseases[J].Tianjin Med J,2014,42(9):946-949.doi:10.3969/j.issn.0253-9896.2014.09.028.
[9]王彦,曹洁,杨庆婵,等.间歇低氧合并肺气肿大鼠系统与内皮炎症状态及外周血内皮祖细胞水平研究[J].天津医药,2014,42(5):427-431.Wang Y,Cao J,Yang QC,et al.Systematic and endothelial inflammation status and endothelial progenitor celllevels in peripheral blood in intermittent hypoxia and emphysemarat model[J].Tianjin Med J,2014,42(5):427-431.doi:10.3969/j.issn.0253-9896.2014.05.008.
[10]Kono M,Tatsumi K,Saibara T,et al.Obstructive sleep apneasyndrome is associated with some components of metabolicsyndrome[J].Chest,2007,131(5):1387-1392.doi:10.1378/chest.06-1807.
[11]Daurat A,Sarhane M,Tiberge M.Obstructive sleep apneasyndrome and cognition:A review[J].Neurophysiol Clin,2016,46(3):201-215.doi:10.1016/j.neucli.2016.04.002.
[12]陈宝元,何权瀛.阻塞性睡眠呼吸暂停综合征的系统性损害[J].中华医学杂志,2012,92(18):1225-1227.Chen BY,He QY.Systemic damage of obstructive sleep apnea syndrome[J].NationalMedical Journal of China,2012,92(18):1225-1227.doi:10.3760/cma.j.issn.0376-2491.2012.18.001.
[13]Almendros I,Khalyfa A,Trzepizur W,et al.Tumor cell malignantproperties are enhanced by circulating exosomes in sleep apnea[J].Chest,2016,150(5):1030-1041.doi:10.1016/j.chest.2016.08.1438.
[14]陈哲,何振华.氧化应激与肺纤维化[J].航空航天医药,2010,21(2):271-273.Chen Z,He ZH.Oxidative stress and pulmonaryfibrosis[J].Aerospace Medicine,2010,21(2):271-273.doi:10.3969/j.issn.2095-1434.2010.02.098.
[15]Yu WN,Sun LF,Yang H.Inhibitory effects of astragaloside IV onbleomycin-induced pulmonary fibrosis in rats via attenuation ofoxidative stress and inflammation[J].Inflammation,2016,39(5):1835-1841.doi:10.1007/s10753-016-0420-5.
[16]王瑞芬,黄坊,孙和国,等.α-SMA和MMP-9在乳腺癌中的表达及意义[J].诊断病理学杂志,2013,20(2):91-93,96.Wang RF,Huang F,Sun HG,et al.Expression ofα-SMA and MMP-9and their clinical significance in breast cancer[J].J Diag Pathol,2013,20(2):91-93,96.doi:10.3969/j.issn.1007-8096.2013.02.008.
[17]谢盛彬,王伟铭,陈楠.UUO模型大鼠肾间质纤维化动态进展及α-SMA、TGF-β1和VDR表达变化[J].上海交通大学学报(医学版),2010,30(7):752-757,796.Xie SB,Wang WM,ChenN.Progression of renal tubulointerstitial fibrosis and expression ofα-SMA,TGF-β1and VDR in rat UUO models[J].Journal ofShanghai Jiao Tong University(Medical Science),2010,30(7):752-757,796.
[18]Ju W,Zhihong Y,Zhiyou Z,et al.Inhibition of alpha-SMA by theectodomain of FGFR2c attenuates lung fibrosis[J].Mol Med,2012,18:992-1002.doi:10.2119/molmed.2011.00425.
[19]Zhang K,Rekhter MD,Gordon D,et al.Myofibroblasts and theirrole in lung collagen gene expression during pulmonary fibrosis.Acombined immunohistochemical and in situ hybridization study[J].Am J Pathol,1994,145(1):114-125.
[20]Hansen NU,Karsdal MA,Brockbank S,et al.Tissue turnover ofcollagen typeⅠ,Ⅲand elastin is elevated in the PCLS model ofIPF and can be restored back to vehicle levels using aphosphodiesterase inhibitor[J].Respir Res,2016,17(1):76.doi:10.1186/s12931-016-0394-8.
[21]Schuliga MJ,See I,Ong SC,et al.Fibrillar collagen clamps lungmesenchymal cells in a nonproliferative and noncontractilephenotype[J].Am J Respir Cell Mol Biol,2009,41(6):731-741.doi:10.1021/bi982966h.
[2]黄绍光,李庆云.睡眠呼吸暂停低通气综合征的流行病学研究现状[J].中华全科医师杂志,2005,4(4):197-198.Huang SG,Li QY.Current status in epidemiological studies on sleep apnea-hypopnea syndrome[J].Chinese Joural of General Practitioners,2005,4(4):197-198.doi:10.3760/cma.j.issn.1671-7368.2005.04.001.
[3]Shamsuzzaman AS,Gersh BJ,Somers VK.Structive sleep apnea:implications for cardiac and vascular disease[J].JAMA,2003,290(14):1906-1914.doi:10.1001/jama.290.14.1906.
[4]Passali D,Corallo G,Yaremchuk S,et al.Oxidative stress inpatients with obstructive sleep apnoea syndrome[J].ActaOtorhinolaryngol Ital,2015,35(6):420-425.doi:10.14639/0392-100X-895.
[5]O’Dwyer DN,Ashley SL,Moore BB.Influences of innate immunity,autophagy,and fibroblast activation in the pathogenesis of lungfibrosis[J].Am J Physiol Lung Cell Mol Physio,2016,311(3):L590-601.doi:10.1152/ajplung.00221.2016.
[6]King TE Jr,Albera C,Bradford WZ,et al.Effect of interferongamma-1b on survival in patients with idiopathic pulmonaryfibrosis(INSPIRE):a multicentre,randomised,placebo-controlledtrial[J].Lancet,2009,374(9685):222-228.doi:10.1016/S0140-6736(09)60551-1.
[7]O’Halloran KD.Chronic intermittent hypoxia creates the perfectstorm with calamitous consequences for respiratory control[J].Respir Physiol Neurobiol,2016,226:63-67.doi:10.1016/j.resp.2015.10.013.
[8]韩苗苗,何庆,施遥,等.睡眠呼吸暂停模式间歇低氧对心脑血管的影响[J].天津医药,2014,42(9):946-949.Han MM,He Q,Shi Y,et al.The impact of intermittent hypoxia from obstructivesleep apnea on cardiovascular and cerebrovascular diseases[J].Tianjin Med J,2014,42(9):946-949.doi:10.3969/j.issn.0253-9896.2014.09.028.
[9]王彦,曹洁,杨庆婵,等.间歇低氧合并肺气肿大鼠系统与内皮炎症状态及外周血内皮祖细胞水平研究[J].天津医药,2014,42(5):427-431.Wang Y,Cao J,Yang QC,et al.Systematic and endothelial inflammation status and endothelial progenitor celllevels in peripheral blood in intermittent hypoxia and emphysemarat model[J].Tianjin Med J,2014,42(5):427-431.doi:10.3969/j.issn.0253-9896.2014.05.008.
[10]Kono M,Tatsumi K,Saibara T,et al.Obstructive sleep apneasyndrome is associated with some components of metabolicsyndrome[J].Chest,2007,131(5):1387-1392.doi:10.1378/chest.06-1807.
[11]Daurat A,Sarhane M,Tiberge M.Obstructive sleep apneasyndrome and cognition:A review[J].Neurophysiol Clin,2016,46(3):201-215.doi:10.1016/j.neucli.2016.04.002.
[12]陈宝元,何权瀛.阻塞性睡眠呼吸暂停综合征的系统性损害[J].中华医学杂志,2012,92(18):1225-1227.Chen BY,He QY.Systemic damage of obstructive sleep apnea syndrome[J].NationalMedical Journal of China,2012,92(18):1225-1227.doi:10.3760/cma.j.issn.0376-2491.2012.18.001.
[13]Almendros I,Khalyfa A,Trzepizur W,et al.Tumor cell malignantproperties are enhanced by circulating exosomes in sleep apnea[J].Chest,2016,150(5):1030-1041.doi:10.1016/j.chest.2016.08.1438.
[14]陈哲,何振华.氧化应激与肺纤维化[J].航空航天医药,2010,21(2):271-273.Chen Z,He ZH.Oxidative stress and pulmonaryfibrosis[J].Aerospace Medicine,2010,21(2):271-273.doi:10.3969/j.issn.2095-1434.2010.02.098.
[15]Yu WN,Sun LF,Yang H.Inhibitory effects of astragaloside IV onbleomycin-induced pulmonary fibrosis in rats via attenuation ofoxidative stress and inflammation[J].Inflammation,2016,39(5):1835-1841.doi:10.1007/s10753-016-0420-5.
[16]王瑞芬,黄坊,孙和国,等.α-SMA和MMP-9在乳腺癌中的表达及意义[J].诊断病理学杂志,2013,20(2):91-93,96.Wang RF,Huang F,Sun HG,et al.Expression ofα-SMA and MMP-9and their clinical significance in breast cancer[J].J Diag Pathol,2013,20(2):91-93,96.doi:10.3969/j.issn.1007-8096.2013.02.008.
[17]谢盛彬,王伟铭,陈楠.UUO模型大鼠肾间质纤维化动态进展及α-SMA、TGF-β1和VDR表达变化[J].上海交通大学学报(医学版),2010,30(7):752-757,796.Xie SB,Wang WM,ChenN.Progression of renal tubulointerstitial fibrosis and expression ofα-SMA,TGF-β1and VDR in rat UUO models[J].Journal ofShanghai Jiao Tong University(Medical Science),2010,30(7):752-757,796.
[18]Ju W,Zhihong Y,Zhiyou Z,et al.Inhibition of alpha-SMA by theectodomain of FGFR2c attenuates lung fibrosis[J].Mol Med,2012,18:992-1002.doi:10.2119/molmed.2011.00425.
[19]Zhang K,Rekhter MD,Gordon D,et al.Myofibroblasts and theirrole in lung collagen gene expression during pulmonary fibrosis.Acombined immunohistochemical and in situ hybridization study[J].Am J Pathol,1994,145(1):114-125.
[20]Hansen NU,Karsdal MA,Brockbank S,et al.Tissue turnover ofcollagen typeⅠ,Ⅲand elastin is elevated in the PCLS model ofIPF and can be restored back to vehicle levels using aphosphodiesterase inhibitor[J].Respir Res,2016,17(1):76.doi:10.1186/s12931-016-0394-8.
[21]Schuliga MJ,See I,Ong SC,et al.Fibrillar collagen clamps lungmesenchymal cells in a nonproliferative and noncontractilephenotype[J].Am J Respir Cell Mol Biol,2009,41(6):731-741.doi:10.1021/bi982966h.