HIF-1a调控VEGF对离体原代培养的大鼠肺动脉平滑肌细胞增殖的影响
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摘要
背景:氧感受器:据知低氧诱导因子1(HIF-1)是细胞适应低氧的关键转录因子,在低氧性肺动脉高压(HPH)也起十分重要的作用,它由氧敏感的α亚基(HIF-1α)和在核内稳定表达的β亚基(HIF-1β)组成的异二聚体转录因子。HIF-1α作为是功能性亚基,可以调控100多种涉及低氧应激下细胞适应与存活的靶基因的表达,从而在低氧应答反应中起核心作用,因而也称为是最新一种的氧感受器。是低氧性肺血管重塑(HPSR)形成中调控目标基因表达的“分子开关”。低氧诱导因子脯氨酰羟化酶(PHD1)是一种可以使HIF-1α脯氨酸残基羟基化并调节其对氧稳定性的重要的关键性酶;低氧诱导因子抑制因子(FIH)是调节HIF-1α转录激活活性的另外一种关键酶。它们通过氧敏感的方式调节低氧诱导因子活性及其表达。
目的:体外原代培养大鼠肺动脉平滑肌细胞(PASMCs),研究低氧下对大鼠PASMCs增殖的影响以及HIF-1α、PHD1、FIH、及HIF-1α靶基因血管内皮生长因子(VEGF)在肺动脉平滑肌细胞中的表达变化的规律及相互关系;观察HIF-1a基因过表达和沉默对PASMC中VEGF的表达的影响,为HPH发病机制提供新的理论依据。
方法:实验共分两部分。第一部分,将SD大鼠消毒后剖开胸腹腔,无菌下取出心肺组织,然后分离出肺动脉段,分离大鼠肺动脉中膜层,用组织块贴壁法培养,倒置相差显微镜观察细胞形态、普通免疫组织化学法(SP法)和免疫荧光法进行细胞鉴定。然后将体外培养的大鼠PASMCs,设计常氧组、低氧组H2,H6,H12,H24。用四唑盐(MTT)比色法测各组PASMCs增殖。用逆转录-聚合酶链反应(RT-PCR)法检测各组细胞HIF-1α、PHD1、FIH、VEGF mRNA表达水平,Western blotting法检测HIF-1α、PHD1、FIH、VEGF的蛋白表达水平。第二部分,将HIF-1a基因转染在大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)中,将细胞分为过表达HIF-1α组、对照组(未转染组)和沉默HIF-1α组。经常氧和低氧处理后分别用Western blotting法检测HIF-1α和VEGF的蛋白表达水平。
结果:(1)在不使用显微技术情况下,用组织贴块法成功培养大鼠PASMCs,得到稳定生长、纯度高的PASMCs,且培养与纯化可以同时进行,可获得稳定传代的细胞。
(2)采用MTT法发现,低氧2小时后肺动脉平滑肌细胞明显增殖,低氧24小时后细胞增殖达高峰,此后增殖稍下降。HIF-1αmRNA和HIF-1α蛋白在常氧组表达阳性,低氧2小时后表达明显增高,低氧6小时后达高峰,此后表达稍下降。PHD1 mRNA和蛋白在常氧组均呈阳性表达,在低氧后表达下降。FIH mRNA和蛋白在常氧组均呈阳性表达,在低氧后表达下降。VEGF mRNA和蛋白在对照组表达少量,低氧2小时后表达升高,低氧12小时后达高峰。
(3)在常氧下siRNA成功沉默HIF-1a基因后(siRNA-HIF-1a组),PASMCs中HIF-1a和VEGF蛋白表达明显减少,对照组(未转染组)表达较siRNA-HIF-1a组增多,过表达HIF-1a组HIF-1a蛋白表达明显增加;在低氧下,siRNA-HIF-1a组HIF-1A和VEGF蛋白仅少量表达,对照组开始增加,过表达组明显增加,且低氧下各组HIF-1a和VEGF蛋白表达较常氧下各组均高。
(4)直线相关分析结果表明:在低氧大鼠肺肺动脉平滑肌细胞HIF-1αmRNA及蛋白与VEGF mRNA、VEGF蛋白均呈正相关。HIF-1α、VEGF蛋白与平滑肌细胞增殖正相关。FIH、PHD1mRNA及蛋白与VEGF mRNA、VEGF蛋白均呈负相关。FIH、PHD1蛋白及mRNA与HIF-1a mRNA、HIF-1a蛋白均呈负相关。
结论:(1)低氧能促进肺动脉平滑肌细胞的低氧性增殖。(2)低氧可诱导肺动脉平滑肌细胞HIF-1a的表达。(3)HIF-1α、VEGF均参与肺动脉平滑肌细胞的增殖过程,HIF-1α可能以转录激活的形式上调VEGF表达,导致平滑肌细胞的增殖。(4)在低氧性肺动脉平滑肌细胞中,PHD1及FIH表达下降,从而减弱了PHD1和FIH对HIF-1α转录激活活性的抑制,导致VEGF等HIF-1α靶基因转录激活,促使肺动脉平滑肌细胞的增殖。
Background Oxygen sensor: hypoxia-inducible factor-1 (HIF-1) which plays a crucial role in the cellular response to the stress of hypoxia and play a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH), is a heterodimeric transcription factor composed of an oxygen sensitiveαsubunit (HIF-1α) and a constitutively expressedβsubunit. HIF-1α, as a functional subunit, regulates the expression of more than 100 genes involved in cellular adaptation and survival and then plays a crucial role in the cellular response to the stress of hypoxia. HIF-1αis also a transcriptional regulator which plays a key role during the development of hypoxic pulmonary vascular remodeling (HPSR). Prolyl hydroxylase domain-containing proteins (PHD1)is a critical enzyme that regulates transactivational activity of HIF-1αthrough post-translational hydroxylation; The factor-inhibiting HIF-1 ( FIH ) is another critical enzyme that regulates transactivational activity of HIF-1α.they regulate the expression and activity of HIF-1αon hypoxia.
Objective Culture primary pulmonary arteria smooth muscle cell in vitro, Investigate the effect of hypoxia on the proliferation of PASMCs and investigated the expression patterns of HIF-1a, PHD1, FIH and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1α, as well as their relationship to each other in the PASMC of rats; investigate whether overexpressing HIF-1αgene and silencing HIF-1αgene by small interfering (si)RNA technology or influenceγ-VEGF gene expression of rat pulmonary arterial smoothmuscle cells (PASMCs) exposure to hypoxia, So as to provide theoretical basis of mechanisms and remedies for HPH.
Methods The whole experiment was divided into two parts: (1) SD-rats was disinfected, dissected in turn. Then lung was catched from chest under aseptic condition. Pulmonary artery was separated and pulmonary artery tissue was planted with the adherent method of tissue explants. The cellular morphology and their typical growth condition were observed with inverted phase contrast microscope. Morphology of the isolated cells were observed by phase-contrast microscopy and identified by immunocytochemistry and immunofluorescence assay using smooth muscle-α-actin antibody.。Cultured rat PASMC were divided into normoxic group; hypoxic group:H2、H6、H12、H24。MTT analysis was used to measure the PASMCs proliferatio;Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of HIF-1a, PHD1, FIH and vascular endothelial growth factor (VEGF),Westen blotting was used to measure protein expression of HIF-1a, PHD1, FIH and VEGF.(2) Transfect HIF-1a in pulmonary arterial smoothmuscle cells (PASMCs),after that the PASMCs were divided into HIF-1a overexpression in transfection group、control group、and siRNA-HIF-1a group.Measure protein expression of HIF-1a, and VEGF after cells exposure to normoxia and hypoxia.
Results (1)Under no micro engineering factor, the primary culture PASMCs with the adherent method of tissue explants can stably grow. Culture and purification can perform in the same time(.2).By MTT, we found that the proliferation of PASMCs in hypoxia increased significantly after 2 h of hypoxia, reached its peak after 24 h of hypoxia, and then declined. HIF-1αmRNA and HIF-1α? protein are poor positively stained in normoxic group and increased dramatically after 2 h of hypoxia, reached its peak after 6 h of hypoxia, and then declined. PHD1 mRNA and protein were poor positively stained in normoxic group, declined when exposure to hypoxia. FIH mRNA and protein were also poor positively stained in normoxic group, declined when exposure to hypoxia. VEGF mRNA and protein were poor positively stained in control, increased markedly after 2 h of hypoxia, and then reached their peak after 12 h of exposure to hypoxia.(3) In normoxic: under the condition of HIF-1a gene silence(siRNA-HIF-1a group),HIF-1 and VEGF protein levels of PASMCs decreased significantly comparing with that in control group(the nontransfected group), increased markedly in HIF-1a overexpression group;In hypoxia, HIF-1 and VEGF protein were poor positively stained in siRNA-HIF-1a group, increased in control group(the nontransfected group), increased markedly in HIF-1a overexpression group. , wherea,HIF-1 and VEGF protein levels of all groups in hypoxia are higher in than normoxic.(4)Linear correlation analysis showed that HIF-1αmRNA and HIF-1αprotein are positively correlated with VEGF mRNA and VEGF protein.HIF-1αprotein and VEGF protein are positively correlated with the proliferation of PAMSC. while PHD1 mRNA、FIH mRNA、PHD1 protein and FIH protein are negatively correlated with VEGF mRNA and VEGF protein. PHD1 mRNA、FIH mRNA、PHD1 protein and FIH protein are negatively correlated with HIF-1a mRNA and HIF-1a protein.
Conclusion (1)Hypoxia stimulates the proliferation of PASMCs.(2)Hypoxia induces the expression of HIF-1 a in PASMCs.(3) Both HIF-1αand VEGF are involved in the proliferation of PASMCs. HIF-1αmay up-regulate the expression of VEGF via transactivation, resulting in the proliferation of PASMCs.(4) The decreased PHD1 and FIH in PASMCs under hypoxia may attenuate its inhibitory effect on the transactivational activity of HIF-1α, promoting the transactivation of HIF-1αtarget genes such as VEGF, and being thus implicated in the proliferation of PASMCs.
目的:体外原代培养大鼠肺动脉平滑肌细胞(PASMCs),研究低氧下对大鼠PASMCs增殖的影响以及HIF-1α、PHD1、FIH、及HIF-1α靶基因血管内皮生长因子(VEGF)在肺动脉平滑肌细胞中的表达变化的规律及相互关系;观察HIF-1a基因过表达和沉默对PASMC中VEGF的表达的影响,为HPH发病机制提供新的理论依据。
方法:实验共分两部分。第一部分,将SD大鼠消毒后剖开胸腹腔,无菌下取出心肺组织,然后分离出肺动脉段,分离大鼠肺动脉中膜层,用组织块贴壁法培养,倒置相差显微镜观察细胞形态、普通免疫组织化学法(SP法)和免疫荧光法进行细胞鉴定。然后将体外培养的大鼠PASMCs,设计常氧组、低氧组H2,H6,H12,H24。用四唑盐(MTT)比色法测各组PASMCs增殖。用逆转录-聚合酶链反应(RT-PCR)法检测各组细胞HIF-1α、PHD1、FIH、VEGF mRNA表达水平,Western blotting法检测HIF-1α、PHD1、FIH、VEGF的蛋白表达水平。第二部分,将HIF-1a基因转染在大鼠肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells, PASMCs)中,将细胞分为过表达HIF-1α组、对照组(未转染组)和沉默HIF-1α组。经常氧和低氧处理后分别用Western blotting法检测HIF-1α和VEGF的蛋白表达水平。
结果:(1)在不使用显微技术情况下,用组织贴块法成功培养大鼠PASMCs,得到稳定生长、纯度高的PASMCs,且培养与纯化可以同时进行,可获得稳定传代的细胞。
(2)采用MTT法发现,低氧2小时后肺动脉平滑肌细胞明显增殖,低氧24小时后细胞增殖达高峰,此后增殖稍下降。HIF-1αmRNA和HIF-1α蛋白在常氧组表达阳性,低氧2小时后表达明显增高,低氧6小时后达高峰,此后表达稍下降。PHD1 mRNA和蛋白在常氧组均呈阳性表达,在低氧后表达下降。FIH mRNA和蛋白在常氧组均呈阳性表达,在低氧后表达下降。VEGF mRNA和蛋白在对照组表达少量,低氧2小时后表达升高,低氧12小时后达高峰。
(3)在常氧下siRNA成功沉默HIF-1a基因后(siRNA-HIF-1a组),PASMCs中HIF-1a和VEGF蛋白表达明显减少,对照组(未转染组)表达较siRNA-HIF-1a组增多,过表达HIF-1a组HIF-1a蛋白表达明显增加;在低氧下,siRNA-HIF-1a组HIF-1A和VEGF蛋白仅少量表达,对照组开始增加,过表达组明显增加,且低氧下各组HIF-1a和VEGF蛋白表达较常氧下各组均高。
(4)直线相关分析结果表明:在低氧大鼠肺肺动脉平滑肌细胞HIF-1αmRNA及蛋白与VEGF mRNA、VEGF蛋白均呈正相关。HIF-1α、VEGF蛋白与平滑肌细胞增殖正相关。FIH、PHD1mRNA及蛋白与VEGF mRNA、VEGF蛋白均呈负相关。FIH、PHD1蛋白及mRNA与HIF-1a mRNA、HIF-1a蛋白均呈负相关。
结论:(1)低氧能促进肺动脉平滑肌细胞的低氧性增殖。(2)低氧可诱导肺动脉平滑肌细胞HIF-1a的表达。(3)HIF-1α、VEGF均参与肺动脉平滑肌细胞的增殖过程,HIF-1α可能以转录激活的形式上调VEGF表达,导致平滑肌细胞的增殖。(4)在低氧性肺动脉平滑肌细胞中,PHD1及FIH表达下降,从而减弱了PHD1和FIH对HIF-1α转录激活活性的抑制,导致VEGF等HIF-1α靶基因转录激活,促使肺动脉平滑肌细胞的增殖。
Background Oxygen sensor: hypoxia-inducible factor-1 (HIF-1) which plays a crucial role in the cellular response to the stress of hypoxia and play a pivotal role during the development of hypoxia-induced pulmonary hypertension (HPH), is a heterodimeric transcription factor composed of an oxygen sensitiveαsubunit (HIF-1α) and a constitutively expressedβsubunit. HIF-1α, as a functional subunit, regulates the expression of more than 100 genes involved in cellular adaptation and survival and then plays a crucial role in the cellular response to the stress of hypoxia. HIF-1αis also a transcriptional regulator which plays a key role during the development of hypoxic pulmonary vascular remodeling (HPSR). Prolyl hydroxylase domain-containing proteins (PHD1)is a critical enzyme that regulates transactivational activity of HIF-1αthrough post-translational hydroxylation; The factor-inhibiting HIF-1 ( FIH ) is another critical enzyme that regulates transactivational activity of HIF-1α.they regulate the expression and activity of HIF-1αon hypoxia.
Objective Culture primary pulmonary arteria smooth muscle cell in vitro, Investigate the effect of hypoxia on the proliferation of PASMCs and investigated the expression patterns of HIF-1a, PHD1, FIH and vascular endothelial growth factor (VEGF), a well-characterized target gene of HIF-1α, as well as their relationship to each other in the PASMC of rats; investigate whether overexpressing HIF-1αgene and silencing HIF-1αgene by small interfering (si)RNA technology or influenceγ-VEGF gene expression of rat pulmonary arterial smoothmuscle cells (PASMCs) exposure to hypoxia, So as to provide theoretical basis of mechanisms and remedies for HPH.
Methods The whole experiment was divided into two parts: (1) SD-rats was disinfected, dissected in turn. Then lung was catched from chest under aseptic condition. Pulmonary artery was separated and pulmonary artery tissue was planted with the adherent method of tissue explants. The cellular morphology and their typical growth condition were observed with inverted phase contrast microscope. Morphology of the isolated cells were observed by phase-contrast microscopy and identified by immunocytochemistry and immunofluorescence assay using smooth muscle-α-actin antibody.。Cultured rat PASMC were divided into normoxic group; hypoxic group:H2、H6、H12、H24。MTT analysis was used to measure the PASMCs proliferatio;Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the mRNA expression of HIF-1a, PHD1, FIH and vascular endothelial growth factor (VEGF),Westen blotting was used to measure protein expression of HIF-1a, PHD1, FIH and VEGF.(2) Transfect HIF-1a in pulmonary arterial smoothmuscle cells (PASMCs),after that the PASMCs were divided into HIF-1a overexpression in transfection group、control group、and siRNA-HIF-1a group.Measure protein expression of HIF-1a, and VEGF after cells exposure to normoxia and hypoxia.
Results (1)Under no micro engineering factor, the primary culture PASMCs with the adherent method of tissue explants can stably grow. Culture and purification can perform in the same time(.2).By MTT, we found that the proliferation of PASMCs in hypoxia increased significantly after 2 h of hypoxia, reached its peak after 24 h of hypoxia, and then declined. HIF-1αmRNA and HIF-1α? protein are poor positively stained in normoxic group and increased dramatically after 2 h of hypoxia, reached its peak after 6 h of hypoxia, and then declined. PHD1 mRNA and protein were poor positively stained in normoxic group, declined when exposure to hypoxia. FIH mRNA and protein were also poor positively stained in normoxic group, declined when exposure to hypoxia. VEGF mRNA and protein were poor positively stained in control, increased markedly after 2 h of hypoxia, and then reached their peak after 12 h of exposure to hypoxia.(3) In normoxic: under the condition of HIF-1a gene silence(siRNA-HIF-1a group),HIF-1 and VEGF protein levels of PASMCs decreased significantly comparing with that in control group(the nontransfected group), increased markedly in HIF-1a overexpression group;In hypoxia, HIF-1 and VEGF protein were poor positively stained in siRNA-HIF-1a group, increased in control group(the nontransfected group), increased markedly in HIF-1a overexpression group. , wherea,HIF-1 and VEGF protein levels of all groups in hypoxia are higher in than normoxic.(4)Linear correlation analysis showed that HIF-1αmRNA and HIF-1αprotein are positively correlated with VEGF mRNA and VEGF protein.HIF-1αprotein and VEGF protein are positively correlated with the proliferation of PAMSC. while PHD1 mRNA、FIH mRNA、PHD1 protein and FIH protein are negatively correlated with VEGF mRNA and VEGF protein. PHD1 mRNA、FIH mRNA、PHD1 protein and FIH protein are negatively correlated with HIF-1a mRNA and HIF-1a protein.
Conclusion (1)Hypoxia stimulates the proliferation of PASMCs.(2)Hypoxia induces the expression of HIF-1 a in PASMCs.(3) Both HIF-1αand VEGF are involved in the proliferation of PASMCs. HIF-1αmay up-regulate the expression of VEGF via transactivation, resulting in the proliferation of PASMCs.(4) The decreased PHD1 and FIH in PASMCs under hypoxia may attenuate its inhibitory effect on the transactivational activity of HIF-1α, promoting the transactivation of HIF-1αtarget genes such as VEGF, and being thus implicated in the proliferation of PASMCs.
引文
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[28] Lando D, Peet DJ, Gorman JJ, et al. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor[J]. Genes Dev, 2002, 16(12): 1466-1471.
[29] Carbia-Nagashima A, Gerez J, Perez-Castro C, et al. RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia[J].Cell,2007,131(2):309-323
[30]吕平,申海涛,张浩,等.消化法培养大鼠脑血管平滑肌细胞[J].中国药理学通报, 2007, 23(2): 272-274.
[31]马丽,刘苏健,邓勇志,等.大鼠胸主动脉平滑肌细胞的培养与鉴定[J].中国心血管病研究杂志, 2007, 5(5): 363-365.
[32] Bur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilir.ation or DNA binding ability of hypoxia-inducible factor-1 a. Mol Pharmacol, 2001, 59 (5):1216-1224
[33] Stenmark KR, Davie NJ, Reeves JT, et al. Hypoxia, leukocytes, and the pulmonary circulation[J].J Appl Physiol, 2005, 98(2): 715-721.
[34]王迪洱。肺动脉高压。王迪得主编,病理生理学。北京:人民卫生出版社,1994,534-545
[35]胡瑞成,戴爱国,谭双香。低氧性肺动脉高压发病中肺动脉平滑肌细胞增殖与凋亡变化。南华大学学报医学版,2001,29 (5): 445-448
[36] Ratcliffe PJ, Ebert BL,Firth JD,et al. Oxygen regulated gene expression: erythropoietin as a model systemAidney Int,1997,51(2):514-526
[37] Stenmark KR,Mechan RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol,1997,59:89-144
[38] Yu AY,Frid MG,Shimoda LA,et al.Temporal,spatial,and oxygen-regulated Expression of hypoxia-inducible factor-1 in the lung.Am J Physiol,1998,275(4ptl):L818-1,826
[39] Wiener CM, Booth G, Semenza GL. In vivo expression of mRNAs encoding hypoxia-inducible factor 1[J]. Biochem Biophys Res Commun, 1996, 225(2): 485-488.
[40] Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain[J]. Eur J Neurosci, 1999, 11(12): 4159-4170.
[41] Belaiba RS, Bonello S, Zahringer C, et al. Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells[J]. Mol Biol Cell, 2007, 18(12): 4691-4697.
[42] Chen YR, Dai AG,Hu RC,et al. Differential and reciprocal regulation between hypoxia-inducible factor-alpha subunits and their prolyl hydroxylases in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2006, 38(6): 423-434.
[43]陈云荣,戴爱国,胡瑞成.低氧诱导因子1α与脯氨酰羟化酶相互调控对大鼠低氧性肺动脉高压的作用[J].中华结核和呼吸杂志, 2006, 29(10): 668-673.
[44]孔春初,戴爱国.丝裂原活化蛋白激酶调节缺氧诱导因子1α对大鼠缺氧性肺动脉高压的作用[J].中华结核和呼吸杂志, 2005, 28(5): 328-332.
[45]孔春初,戴爱国.磷酸肌醇3-激酶调控缺氧诱导因子1α对大鼠缺氧性肺动脉高压的作用[J].中国病理生理杂志, 2006, 22(11): 2132-2137.
[46] Li QF, Dai AG. Differential expression of three hypoxia-inducible factor-alpha subunits in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2005, 37(10): 665-672.
[47] Stolze IP, Tian YM, Appelhoff RJ, et al. Genetic analysis of the role of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (HIF) in regulating HIF transcriptional target genes[J]. J Biol Chem, 2004, 279(41): 42719-42725
[48] Huang LE, Willmore WG, Gu J, et al. Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J Biol Chem, 1999, 274(13): 9038-9044.
[1] Lopez-Barneo J, Pardal R, Ortega-Saenz P. Cellular mechanism of oxygen sensing[J]. Annu Rev Physiol, 2001, 63259-287.
[2] Wenger RH. Mammalian oxygen sensing, signalling and gene regulation[J]. J Exp Biol, 2000, 203(Pt 8): 1253-1263.
[3] Li QF, Dai AG. Differential expression of three hypoxia-inducible factor-alpha subunits in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2005, 37(10): 665-672.
[4] Buckler KJ, Vaughan-Jones RD. Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells[J]. J Physiol, 1994, 476(3): 423-428.
[5] Lahiri S. Historical perspectives of cellular oxygen sensing and responses to hypoxia[J]. J Appl Physiol, 2000, 88(4): 1467-1473.
[6] Kemp PJ, Lewis A, Hartness ME, et al. Airway chemotransduction: from oxygen sensor to cellular effector[J]. Am J Respir Crit Care Med, 2002, 166(12 Pt 2): S17-24.
[7] Peers C, Kemp PJ. Acute oxygen sensing: diverse but convergent mechanisms in airway and arterial chemoreceptors[J]. Respir Res, 2001, 2(3): 145-149.
[8] Kemp PJ, Searle GJ, Hartness ME, et al. Acute oxygen sensing in cellular models: relevance to the physiology of pulmonary neuroepithelial and carotid bodies[J]. Anat Rec A Discov Mol Cell Evol Biol, 2003, 270(1): 41-50.
[9] Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein[J]. Science, 1988, 242(4884): 1412-1415.
[10] Duchen MR. Contributions of mitochondria to animal physiology: fromhomeostatic sensor to calcium signalling and cell death[J]. J Physiol, 1999, 516 ( Pt 1)1-17.
[11] Weir EK, Lopez-Barneo J, Buckler KJ, et al. Acute oxygen-sensing mechanisms[J]. N Engl J Med, 2005, 353(19): 2042-2055.
[12] Thompson RJ, Buttigieg J, Zhang M, et al. A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells[J]. Neuroscience, 2007, 145(1): 130-141.
[13] Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing[J]. Circ Res, 2001, 88(12): 1259-1266.
[14] Archer S, Michelakis E. The mechanism(s) of hypoxic pulmonary vasoconstriction: potassium channels, redox O(2) sensors, and controversies[J]. News Physiol Sci, 2002, 17131-137.
[15] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[J]. Physiol Rev, 2007, 87(1): 245-313.
[16] He L, Dinger B, Sanders K, et al. Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells[J]. Am J Physiol Lung Cell Mol Physiol, 2005, 289(6): L916-924.
[17] Wolin MS, Ahmad M, Gupte SA. Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH[J]. Am J Physiol Lung Cell Mol Physiol, 2005, 289(2): L159-173.
[18] Dinger B, He L, Chen J, et al. The role of NADPH oxidase in carotid body arterial chemoreceptors[J]. Respir Physiol Neurobiol, 2007, 157(1): 45-54.
[19] Archer SL, Reeve HL, Michelakis E, et al. O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase[J]. Proc Natl Acad Sci U S A, 1999, 96(14): 7944-7949.
[20] Buckler KJ. A novel oxygen-sensitive potassium current in rat carotid body type I cells[J]. J Physiol, 1997, 498 ( Pt 3)649-662.
[21] Hu RC,Dai AG,Tan SX. Hypoxia-inducible factor 1αupregulate the expression of inducible nitric oxide synthase gene in pulmonary arteries of hypoxic rat. Chin Med J, 2002,115:1833-1837.
[22] Metzen E, Berchner-Pfannschmidt U, Stengel P, et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing[J]. J Cell Sci, 2003, 116(Pt 7): 1319-1326.
[23] Ehrismann D, Flashman E, Genn DN, et al. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay[J]. Biochem J, 2007, 401(1): 227-234.
[24] Dalgard CL, Lu H, Mohyeldin A, et al. Endogenous 2-oxoacids differentially regulate expression of oxygen sensors[J]. Biochem J, 2004, 380(Pt 2): 419-424.
[25] Srinivas V, Zhu X, Salceda S, et al. Hypoxia-inducible factor 1alpha (HIF-1alpha) is a non-heme iron protein. Implications for oxygen sensing[J]. J Biol Chem, 1998, 273(29): 18019-18022.
[26] Kallio PJ, Wilson WJ, O'Brien S, et al. Regulation of the hypoxia-inducible transcription factor 1alpha by the ubiquitin-proteasome pathway[J]. J Biol Chem, 1999, 274(10): 6519-6525.
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[26] Lancaster DE, McNeill LA, McDonough MA, et al. Disruption ofdimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity. Biochem J, 2004, 383(Pt. 3): 429-437.
[27] Koivunen P, Hirsila M, Gunzler V, et al. Catalytic properties of the asparaginyl hydroxylase (FIH) in the oxygen sensing pathway are distinct from those of its prolyl 4-hydroxylases. J Biol Chem, 2004, 279(11): 9899-9904.
[28] Lando D, Peet DJ, Gorman JJ, et al. FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor[J]. Genes Dev, 2002, 16(12): 1466-1471.
[29] Carbia-Nagashima A, Gerez J, Perez-Castro C, et al. RSUME, a small RWD-containing protein, enhances SUMO conjugation and stabilizes HIF-1alpha during hypoxia[J].Cell,2007,131(2):309-323
[30]吕平,申海涛,张浩,等.消化法培养大鼠脑血管平滑肌细胞[J].中国药理学通报, 2007, 23(2): 272-274.
[31]马丽,刘苏健,邓勇志,等.大鼠胸主动脉平滑肌细胞的培养与鉴定[J].中国心血管病研究杂志, 2007, 5(5): 363-365.
[32] Bur E, Chang KY, Lee E, et al. Mitogen-activated protein kinase kinase inhibitor PD98059 blocks the trans-activation but not the stabilir.ation or DNA binding ability of hypoxia-inducible factor-1 a. Mol Pharmacol, 2001, 59 (5):1216-1224
[33] Stenmark KR, Davie NJ, Reeves JT, et al. Hypoxia, leukocytes, and the pulmonary circulation[J].J Appl Physiol, 2005, 98(2): 715-721.
[34]王迪洱。肺动脉高压。王迪得主编,病理生理学。北京:人民卫生出版社,1994,534-545
[35]胡瑞成,戴爱国,谭双香。低氧性肺动脉高压发病中肺动脉平滑肌细胞增殖与凋亡变化。南华大学学报医学版,2001,29 (5): 445-448
[36] Ratcliffe PJ, Ebert BL,Firth JD,et al. Oxygen regulated gene expression: erythropoietin as a model systemAidney Int,1997,51(2):514-526
[37] Stenmark KR,Mechan RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol,1997,59:89-144
[38] Yu AY,Frid MG,Shimoda LA,et al.Temporal,spatial,and oxygen-regulated Expression of hypoxia-inducible factor-1 in the lung.Am J Physiol,1998,275(4ptl):L818-1,826
[39] Wiener CM, Booth G, Semenza GL. In vivo expression of mRNAs encoding hypoxia-inducible factor 1[J]. Biochem Biophys Res Commun, 1996, 225(2): 485-488.
[40] Bergeron M, Yu AY, Solway KE, et al. Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain[J]. Eur J Neurosci, 1999, 11(12): 4159-4170.
[41] Belaiba RS, Bonello S, Zahringer C, et al. Hypoxia up-regulates hypoxia-inducible factor-1alpha transcription by involving phosphatidylinositol 3-kinase and nuclear factor kappaB in pulmonary artery smooth muscle cells[J]. Mol Biol Cell, 2007, 18(12): 4691-4697.
[42] Chen YR, Dai AG,Hu RC,et al. Differential and reciprocal regulation between hypoxia-inducible factor-alpha subunits and their prolyl hydroxylases in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2006, 38(6): 423-434.
[43]陈云荣,戴爱国,胡瑞成.低氧诱导因子1α与脯氨酰羟化酶相互调控对大鼠低氧性肺动脉高压的作用[J].中华结核和呼吸杂志, 2006, 29(10): 668-673.
[44]孔春初,戴爱国.丝裂原活化蛋白激酶调节缺氧诱导因子1α对大鼠缺氧性肺动脉高压的作用[J].中华结核和呼吸杂志, 2005, 28(5): 328-332.
[45]孔春初,戴爱国.磷酸肌醇3-激酶调控缺氧诱导因子1α对大鼠缺氧性肺动脉高压的作用[J].中国病理生理杂志, 2006, 22(11): 2132-2137.
[46] Li QF, Dai AG. Differential expression of three hypoxia-inducible factor-alpha subunits in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2005, 37(10): 665-672.
[47] Stolze IP, Tian YM, Appelhoff RJ, et al. Genetic analysis of the role of the asparaginyl hydroxylase factor inhibiting hypoxia-inducible factor (HIF) in regulating HIF transcriptional target genes[J]. J Biol Chem, 2004, 279(41): 42719-42725
[48] Huang LE, Willmore WG, Gu J, et al. Inhibition of hypoxia-inducible factor 1 activation by carbon monoxide and nitric oxide. Implications for oxygen sensing and signaling. J Biol Chem, 1999, 274(13): 9038-9044.
[1] Lopez-Barneo J, Pardal R, Ortega-Saenz P. Cellular mechanism of oxygen sensing[J]. Annu Rev Physiol, 2001, 63259-287.
[2] Wenger RH. Mammalian oxygen sensing, signalling and gene regulation[J]. J Exp Biol, 2000, 203(Pt 8): 1253-1263.
[3] Li QF, Dai AG. Differential expression of three hypoxia-inducible factor-alpha subunits in pulmonary arteries of rat with hypoxia-induced hypertension[J]. Acta Biochim Biophys Sin (Shanghai), 2005, 37(10): 665-672.
[4] Buckler KJ, Vaughan-Jones RD. Effects of hypoxia on membrane potential and intracellular calcium in rat neonatal carotid body type I cells[J]. J Physiol, 1994, 476(3): 423-428.
[5] Lahiri S. Historical perspectives of cellular oxygen sensing and responses to hypoxia[J]. J Appl Physiol, 2000, 88(4): 1467-1473.
[6] Kemp PJ, Lewis A, Hartness ME, et al. Airway chemotransduction: from oxygen sensor to cellular effector[J]. Am J Respir Crit Care Med, 2002, 166(12 Pt 2): S17-24.
[7] Peers C, Kemp PJ. Acute oxygen sensing: diverse but convergent mechanisms in airway and arterial chemoreceptors[J]. Respir Res, 2001, 2(3): 145-149.
[8] Kemp PJ, Searle GJ, Hartness ME, et al. Acute oxygen sensing in cellular models: relevance to the physiology of pulmonary neuroepithelial and carotid bodies[J]. Anat Rec A Discov Mol Cell Evol Biol, 2003, 270(1): 41-50.
[9] Goldberg MA, Dunning SP, Bunn HF. Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein[J]. Science, 1988, 242(4884): 1412-1415.
[10] Duchen MR. Contributions of mitochondria to animal physiology: fromhomeostatic sensor to calcium signalling and cell death[J]. J Physiol, 1999, 516 ( Pt 1)1-17.
[11] Weir EK, Lopez-Barneo J, Buckler KJ, et al. Acute oxygen-sensing mechanisms[J]. N Engl J Med, 2005, 353(19): 2042-2055.
[12] Thompson RJ, Buttigieg J, Zhang M, et al. A rotenone-sensitive site and H2O2 are key components of hypoxia-sensing in neonatal rat adrenomedullary chromaffin cells[J]. Neuroscience, 2007, 145(1): 130-141.
[13] Waypa GB, Chandel NS, Schumacker PT. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing[J]. Circ Res, 2001, 88(12): 1259-1266.
[14] Archer S, Michelakis E. The mechanism(s) of hypoxic pulmonary vasoconstriction: potassium channels, redox O(2) sensors, and controversies[J]. News Physiol Sci, 2002, 17131-137.
[15] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[J]. Physiol Rev, 2007, 87(1): 245-313.
[16] He L, Dinger B, Sanders K, et al. Effect of p47phox gene deletion on ROS production and oxygen sensing in mouse carotid body chemoreceptor cells[J]. Am J Physiol Lung Cell Mol Physiol, 2005, 289(6): L916-924.
[17] Wolin MS, Ahmad M, Gupte SA. Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH[J]. Am J Physiol Lung Cell Mol Physiol, 2005, 289(2): L159-173.
[18] Dinger B, He L, Chen J, et al. The role of NADPH oxidase in carotid body arterial chemoreceptors[J]. Respir Physiol Neurobiol, 2007, 157(1): 45-54.
[19] Archer SL, Reeve HL, Michelakis E, et al. O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase[J]. Proc Natl Acad Sci U S A, 1999, 96(14): 7944-7949.
[20] Buckler KJ. A novel oxygen-sensitive potassium current in rat carotid body type I cells[J]. J Physiol, 1997, 498 ( Pt 3)649-662.
[21] Hu RC,Dai AG,Tan SX. Hypoxia-inducible factor 1αupregulate the expression of inducible nitric oxide synthase gene in pulmonary arteries of hypoxic rat. Chin Med J, 2002,115:1833-1837.
[22] Metzen E, Berchner-Pfannschmidt U, Stengel P, et al. Intracellular localisation of human HIF-1 alpha hydroxylases: implications for oxygen sensing[J]. J Cell Sci, 2003, 116(Pt 7): 1319-1326.
[23] Ehrismann D, Flashman E, Genn DN, et al. Studies on the activity of the hypoxia-inducible-factor hydroxylases using an oxygen consumption assay[J]. Biochem J, 2007, 401(1): 227-234.
[24] Dalgard CL, Lu H, Mohyeldin A, et al. Endogenous 2-oxoacids differentially regulate expression of oxygen sensors[J]. Biochem J, 2004, 380(Pt 2): 419-424.
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