CAV1在人肺动脉成纤维细胞增殖调控中的作用
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摘要
目的研究小窝蛋白1(CAV1)在缺氧相关肺动脉成纤维细胞(PAFs)增殖调控中的作用。方法将体外培养的人PAFs分为:对照组(C:21%O_2);10%氧浓度组(10%O_2);5%氧浓度组(5%O_2)及2%氧浓度组(2%O_2)进行细胞增殖实验,选取细胞增殖最明显组为缺氧组(H)进行后续实验。并构建CAV1高表达质粒(pCAV1),然后再将PAFs分为对照组(C:21%O_2),缺氧组(H),空白对照组(NC:缺氧+空质粒转染组)和CAV1高表达组(pCAV1:缺氧+pCAV1转染组),采用Western-blot法检测各组细胞中CAV1、细胞周期蛋白D1(cyclinD1)和细胞凋亡抑制蛋白2(c-IAP2)含量,四甲基偶氮唑蓝(MTT)及增殖细胞核抗原(PCNA)免疫组化法检测细胞增殖情况。结果缺氧能刺激PAFs增殖,并呈浓度依赖性,于2%氧浓度刺激48小时PAFs增殖达峰值,与对照组相比差异有统计学意义(1.20+0.02 vs 0.54+0.04,P<0.01);缺氧组PAFs中CAV1表达下调(1.23±0.04 vs 0.90±0.02,P<0.01),cyclin D1(0.19±0.03 vs 1.15±0.06,P<0.01)和c-IAP2(0.63±0.04 vs 0.78±0.09,P<0.01)表达上调,细胞增殖增加(MTT:0.78±0.04 vs 1.20±0.02,P<0.01;PCNA:0.29±0.03 vs 0.54±0.03,P<0.01);CAV1在PAFs中高表达后(0.55±0.03 vs 0.90±0.03,P<0.01),cyclin D1(0.88±0.02 vs 0.52±0.02,P<0.01)和c-IAP2(0.87±0.02 vs 0.72±0.02,P<0.01)表达下调,PAFs增殖减少(MTT:1.20±0.02 vs 1.00±0.06,P<0.01;PCNA:0.52±0.03 vs 0.38±0.03,P<0.01)。结论缺氧能通过下调CAV-1在PAFs中的表达,促进PAFs的增殖、抑制其凋亡。cyclinD1和cIAP2可能是CAV1调控PAFs增殖和凋亡的下游靶点。
Objective To study the role of CAV1 in hypoxia associated proliferation of human pulmonary artery fibroblasts(PAFs). Methods The PAFs cultured in vitro were divided into four groups,the control group(C:21% O_2),the 10% oxygen group(10% O_2),the 5% oxygen group(5% O_2) and the 2% oxygen group(2% O_2),and then they were given cells proliferation test and the maximal proliferation group was selected as the hypoxia group(H) for further study. The high expression of CAV1 plasmid(pCAV1) was constructed. After that,the PAFs were divided into the control group(C: 21% O_2),the hypoxia group(H),the blank control group(NC: hypoxia + null plasmid group) and the high expression of CAV1 plasmid group(pCAV1),and then the cells proliferation was detected by MTT and PCNA immunohistochemistry,and the expression of CAV1,cyclin D1 and c-IAP2 by western-blot in each group respectively. Results Hypoxia induced the proliferation of PAFs in dose dependent manner,and the maximal PAFs proliferation were observed in the 2% O_2 group within 48 h while compared with the groupC(1. 20 +0. 02 vs 0. 54 + 0. 04,P < 0. 01). In the hypoxia group,the expression of CAV1 decreased(1. 23 ± 0. 04 vs 0. 90 ±0. 02,P < 0. 01),while the expression of cyclin D1(0. 19 ± 0. 03 vs 1. 15 ± 0. 06,P < 0. 01) and c-IAP2(0. 63 ±0. 04 vs 0. 78 ± 0. 09,P < 0. 01) increased,and PAFs proliferation increased(MTT: 0. 78 ± 0. 04 vs 1. 20 ± 0. 02,P< 0. 01; PCNA: 0. 29 ± 0. 03 vs 0. 54 ± 0. 03,P < 0. 01). When CAV1 was high expressed in PAFs(0. 55 ± 0. 03 vs 0. 90 ± 0. 03,P < 0. 01),the expression of cyclin D1(0. 88 ± 0. 02 vs 0. 52 ± 0. 02,P < 0. 01) and c-IAP2(0. 87± 0. 02 vs 0. 72 ± 0. 02,P < 0. 01) decreased,and PAFs proliferation decreased,too(MTT: 1. 20 ± 0. 02 vs 1. 00 ±0. 06,P < 0. 01; PCNA: 0. 52 ± 0. 03 vs 0. 38 ± 0. 03,P < 0. 01). Conclusion Hypoxia can down-regulate CAV1 expression in PAFs,promote PAFs proliferation and inhibit PAFs apoptosis,and cyclin D1 and c-IAP2 may be the downstream targets of CAV1 in the regulation of PAFs proliferation and apoptosis.
Objective To study the role of CAV1 in hypoxia associated proliferation of human pulmonary artery fibroblasts(PAFs). Methods The PAFs cultured in vitro were divided into four groups,the control group(C:21% O_2),the 10% oxygen group(10% O_2),the 5% oxygen group(5% O_2) and the 2% oxygen group(2% O_2),and then they were given cells proliferation test and the maximal proliferation group was selected as the hypoxia group(H) for further study. The high expression of CAV1 plasmid(pCAV1) was constructed. After that,the PAFs were divided into the control group(C: 21% O_2),the hypoxia group(H),the blank control group(NC: hypoxia + null plasmid group) and the high expression of CAV1 plasmid group(pCAV1),and then the cells proliferation was detected by MTT and PCNA immunohistochemistry,and the expression of CAV1,cyclin D1 and c-IAP2 by western-blot in each group respectively. Results Hypoxia induced the proliferation of PAFs in dose dependent manner,and the maximal PAFs proliferation were observed in the 2% O_2 group within 48 h while compared with the groupC(1. 20 +0. 02 vs 0. 54 + 0. 04,P < 0. 01). In the hypoxia group,the expression of CAV1 decreased(1. 23 ± 0. 04 vs 0. 90 ±0. 02,P < 0. 01),while the expression of cyclin D1(0. 19 ± 0. 03 vs 1. 15 ± 0. 06,P < 0. 01) and c-IAP2(0. 63 ±0. 04 vs 0. 78 ± 0. 09,P < 0. 01) increased,and PAFs proliferation increased(MTT: 0. 78 ± 0. 04 vs 1. 20 ± 0. 02,P< 0. 01; PCNA: 0. 29 ± 0. 03 vs 0. 54 ± 0. 03,P < 0. 01). When CAV1 was high expressed in PAFs(0. 55 ± 0. 03 vs 0. 90 ± 0. 03,P < 0. 01),the expression of cyclin D1(0. 88 ± 0. 02 vs 0. 52 ± 0. 02,P < 0. 01) and c-IAP2(0. 87± 0. 02 vs 0. 72 ± 0. 02,P < 0. 01) decreased,and PAFs proliferation decreased,too(MTT: 1. 20 ± 0. 02 vs 1. 00 ±0. 06,P < 0. 01; PCNA: 0. 52 ± 0. 03 vs 0. 38 ± 0. 03,P < 0. 01). Conclusion Hypoxia can down-regulate CAV1 expression in PAFs,promote PAFs proliferation and inhibit PAFs apoptosis,and cyclin D1 and c-IAP2 may be the downstream targets of CAV1 in the regulation of PAFs proliferation and apoptosis.
引文
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[14]Yang DL,Zhang HG,Xu YL,et al.Resveratrol inhibits right ventricular hypertrophy induced by monocrotaline in rats[J].Clin Exp Pharmacol Physiol,2010,37:150-155.
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[2]Stenmark KR,Fagan KA,Frid MG.Hypoxia-induced pulmonary vascular remodeling cellular and molecular mechanisms[J].Circulation research,2006,99(7):675-691.
[3]Chen C,Han X,Fan F,et al.Serotonin drives the activation of pulmonary artery adventitial fibroblasts and TGF-β1/Smad3-mediated fibrotic responses through 5-HT2A receptors[J].Mol Cell Biochem,2014,397(1-2):267-276.
[4]Goetz JG,Lajoie P,Wiseman SM,et al.Caveolin-1 in tumor progression:the good,the bad and the ugly[J].Cancer Metastasis Reviews,2008,27(4):715-735.
[5]Patel HH,Zhang S,Murray F,et al.Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension.[J].FASEB J,2007,21(11):2970-2979.
[6]Bakhshi FR,Mao M,Shajahan AN,et al.Nitrosation-dependent caveolin 1 phosphorylation,ubiquitination,and degradation and its association with idiopathic pulmonary arterial hypertension[J].Pulmonary Circulation,2013,3(4):816-830.
[7]Sehgal PB,Lee JE.Protein trafficking dysfunctions:Role in the pathogenesis of pulmonary arterial hypertension[J].Pulmonary Circulation,2011,1(1):17-32.
[8]Cohen AW,Razani B,Schubert W,et al.Role of caveolin-1 in the modulation of lip-olysis and lipid roplet formation[J].Diabetes,2004,53(5):1261-1270.
[9]Wang XM,Kim HP,Song R,et al.Caveolin-1 confers antiinflammatory effects in murine macrophages via the MKK3/p38MAPK pathway[J].Am J Respir Cell Mol Biol,2006,34(4):434
[10]左蓓,刑敏,孙珍贵,等.低氧诱导的小窝蛋白-1上调参与人肺腺癌细胞A549迁移和侵袭[J].中国病理生理杂志,2014,30(10):1794-1799.
[11]Wang KY,Lee MF,Ho HC,et al.Serum Caveolin-1 as a Novel Biomarker in Idiopathic Pulmonary Artery Hypertension[J].Biomed Res Int,2015,10:1-7.
[12]Cruz JA,Bauer EM,Rodriguez AI,et al.Chronic hypoxia induces right heart failure in caveolin-1-/-mice[J].Am J Physiol Heart Circ Physiol,2012,302(12):H2518-H2527.
[13]王苒,徐永健,刘先胜,等.结缔组织生长因子和周期蛋白D1在烟雾暴露大鼠肺血管重塑中的表达变化[J].中华结核和呼吸杂志,2010,33(9):679-683.
[14]Yang DL,Zhang HG,Xu YL,et al.Resveratrol inhibits right ventricular hypertrophy induced by monocrotaline in rats[J].Clin Exp Pharmacol Physiol,2010,37:150-155.
[15]Kolli MB,Manne ND,Para R,et al.Cerium oxide nanoparticles attenuate monocrotaline induced right ventricular hypertrophy following pulmonary arterial hypertension[J].Biomaterials,2014,35(37):9951-9962.
[16]Augello C,Caruso L,Maggioni M,et al.Inhibitors of apoptosis proteins(IAPs)expression and their prognostic significance in hepatocellular carcinoma[J].BMC Cancer,2009,9(1):1-10.