缺氧对肺动脉内皮细胞环氧合酶,血栓素合成酶基因表达的影响

应用分子杂交技术和放射免疫检测方法研究了缺氧对猪肺动脉内皮细胞的环氧合酶（ＣＯＸ）和血栓素合成酶（ＴＸＳ）基因表达及其条件培养基中６ｋｅｔｏＰＧＦ１α和ＴＸＢ２含量的动态变化。发现：６,１２,２４和４８ｈ缺氧组分别与常氧组比,ＣＯＸ１和ＣＯＸ２基因表达增加,并且ＣＯＸ２ｍＲＮＡ在缺氧６和１２ｈ就明显表达增加。在前述的不同缺氧时间组内皮细胞条件培养基中６ｋｅｔｏＰＧＦ１α含量也均显著高于相应常氧对照组（Ｐ＜０．０５）;但ＴＸＳ的ｍＲＮＡ水平及ＴＸＢ２含量在缺氧４８ｈ才有明显增加（Ｐ＜０．０５）。结果表明：（１）缺氧可诱导肺动脉内皮ＣＯＸ基因表达和ＰＧＩ２生成增加,在早期以ＣＯＸ２基因表达增加更为明显,提示可能在肺血管缺氧反应中起调节作用。（２）４８ｈ的缺氧可使内皮细胞ＴＸＳ基因表达及ＴＸＡ２生成增加,它可能在慢性缺氧肺血管反应中起介导作用。     

缺氧性肺动脉高压大鼠肺组织中血小板源性生长因子和c—my…

本研究分析了大鼠肺组织中血小板源性生长因子Ａ链、Ｂ链和ｃ－ｍｙｃ原癌基因ｍＲＮＡ。正常肺组织可表达１．７ｋｂ的ＰＤＧＦ－ＡｍＲＮＡ和３．５ｋｂ的ＰＤＧＦ－ＢｍＲＮＡ,还有少量２．２ｋｂｃｍＲＮＡ．在缺氧过程中,ＰＤＧＦ－Ｂ链ｍＲＮＡ和ｃ－ｍｙｃｍＲＮＡ迅速增加,至缺氧１４ｄ时,分别为正常的３倍和５倍。而ＰＤＧＦ－ＡｍＲＮＡ在缺氧７ｄ时增高,而后又略有降低。结果表明：缺氧的肺组织局生成的ＰＤＧＦ激活     

慢性缺氧大鼠肺血管c-myc及bcl-2基因表达研究

应用免疫组织化学和Ｎｏｒｔｈｅｒｎ杂交方法,对慢性缺氧大鼠肺组织（主要是肺血管壁）原癌基因ｃ－ｍｙｃ和抗凋亡基因ｂｃｌ－２表达进行了研究。免疫组织化学观察提示,正常对照组大鼠肺血壁Ｃ－ｍｙｃ和Ｂｃｌ－２蛋白仅为弱阳性或不表达,慢性缺氧１、２周组大鼠肺血管壁这两种抗原表达比对照组显著增强,呈强阳性,Ｎｏｒｔｈｅｒｎ杂交结果表明：慢性缺氧１、２周组大鼠肺组织内ｃ－ｍｙｃ和ｂｃｌ－２的ｍＲＮＡ表达比对照组显著增加,以上结果提示,ｃ－ｍｙｃ及ｂｃｌ－２两种基因调节的细胞增殖与凋亡可能参与了慢性缺氧性肺动脉高压的发病进程。     

缺氧性肺动脉高压大鼠肺组织中血小板源性生长因子和c－myc基因表达增强

本研究分析了大鼠肺组织中血小板源性生长因子Ａ链、Ｂ链（ＰＤＧＦ－Ａ,ＰＤＧＦ－Ｂ）和ｃ－ｍｙｃ原癌基因ｍＲＮＡ在正常和缺氧时的含量变化。正常肺组织可表达１．７ｋｂ的ＰＤＧＦ－ＡｍＲＮＡ和３．５ｋｂ的ＰＤＧＦ－ＢｍＲＮＡ,还有少量２．２ｋｂｃ－ｍｙｃｍＲＮＡ。在缺氧过程中,ＰＤＧＦ－Ｂ链ｍＲＮＡ和ｃ－ｍｙｃｍＲＮＡ迅速增加,至缺氧１４ｄ时,分别为正常的３倍和５倍。而ＰＤＧＦ－ＡｍＲＮＡ在缺氧７ｄ时增高,而后又略有降低。结果表明：缺氧的肺组织局部生成的ＰＤＧＦ激活了ｃ－ｍｙｃ原癌基因,这对于缺氧性肺动脉高压的形成具有重要作用。     

缺氧大鼠肺原癌基因jun表达之研究

本文用Ｎｏｒｔｈｅｒｎｂｌｏｔ杂交和原位杂交技术检测了缺氧大鼠肺内原癌基因ｊｕｎ的表达,发现正常大鼠肺脏有一定基础量的ｊｕｎｍＲＮＡ表达,缺氧１Ｗ后肺内ｊｕｎ表达有所下降,缺氧２Ｗ时,ｊｕｎ表达水平又回升,至缺氧第３Ｗ,ｊｕｎｍＲＮＡ之表达明显增加。结果提示缺氧可刺激肺内原癌基因的表达,过度表达的癌基因蛋白可能在缺氧性肺动脉高压的形成过程中具有一定意义。     

缺氧对大鼠肺组织肺血管sis原癌基因和PDGF-BB蛋白表达的影响

为了探讨血小板生长因子ＢＢ（ＰＤＧＦ－ＢＢ）在缺氧性肺血管结构重塑中作用,通过建立大鼠缺氧性肺血管重塑动物模型,运用原位杂交技术和免疫组织化学染色技术检测不同缺氧时期大鼠肺组织中原癌基因ｓｉｓｍＲＮＡ和ＰＤＧＦ－ＢＢ蛋白表达。结果：（１）随着缺氧时间的延长,肺动脉壁肌层增厚,管腔变窄;（２）正常对照组肺组织及肺血管壁ｃ－ｓｉｓｍＲＮＡ和ＰＤＧＦ－ＢＢ蛋白质均极少表达（＋）;（３）缺氧３天后,两者表达均增多（）;（４）缺氧７天后其表达达高峰并持续至１４天（～）;（５）缺氧２１天后,两者表达均降低,但仍高于正常对照组（）。提示：缺氧可以刺激大鼠肺组织ｓｉｓｍＲＮＡ和ＰＤＧＦ－ＢＢ蛋白表达,其在缺氧性肺血管结构重塑形成中可能具有重要作用。     

缺氧和DDPH对人胚肺成纤维细胞前胶原mRNA表达的影响

用核酸原位杂交和图像分析等方法,观察直接缺氧（Ｈ）和缺氧猪肺动脉内皮细胞条件培养液（ＨＥＣＣＭ）对人胚肺成纤维细胞（ＫＭＢ１７）的前胶原ｐｒｏα１（Ⅰ）,ｐｒｏα１（Ⅲ）ｍＲＮＡ表达和抗高血压药１－（２,６－二甲基苯氧基）－２－（３,４－二甲氧基苯乙氨基）丙烷盐酸盐（ＤＤＰＨ）对此过程的影响。结果发现,Ｈ和ＨＥＣＣＭ均可使ＫＭＢ１７的两型前胶原ｍＲＮＡ表达量增高,明显高于对照组（Ｐ＜０．０１）。ＤＤＰＨ对ＨＥＣＣＭ组细胞的Ⅰ,Ⅲ两型前胶原ｍＲＮＡ表达增高均有显著的抑制作用（抑制率分别为－４３．９７％和－５６．２２％）,而对Ｈ组仅抑制ｐｒｏα１（Ⅰ）前胶原ｍＲＮＡ的过量表达（－５３．５８％）。提示缺氧可直接或通过肺动脉内皮细胞的介导,促进人胚肺成纤维细胞的Ⅰ、Ⅲ两型前胶原ｍＲＮＡ表达,ＤＤＰＨ在基因转录水平上对此过程有抑制作用     

烫伤对大鼠下丘脑视上核内皮素-1基因转录和蛋白含量的影响

用原位杂交和免疫组织化学方法观察了烫伤后下丘脑视上核（ＳＯＮ）内皮素－１（ＥＴ－１）基因转录和蛋白含量的变化,并用通用图像颗粒分析法估计ＥＴ－１ ｍＲＮＡ阳性杂交信号的强度和ＥＴ－１样免疫反应物（ＥＴ－１－ｉｒ）的免疫反应强度。与对照组相比,烫伤后１５ｍｉｎ,ＳＯＮ神经元胞浆内ＥＴ－１ ｍＲＮＡ阳性杂交信号未见明显变化;而在烫伤后６０和１８０ｍｉｎ,ＥＴ－１ ｍＲＮＡ阳性杂交信号强度分别较比照组增     

缺氧诱导因子1和红细胞生成素3‘—增强子调节内皮细胞？…

目的和方法：将红细胞生成素（ＥＰＯ）３＇－增强子野生片断及点突变片断借脂质体主人脐静脉内皮细胞株ＥＣＶ－３０４,用半定量ＲＴ－ＰＣＲ测定正常秘缺氧诱导因子－１（ＨＩＦ－１）诱导剂氧化钴（ＣｏＣｌ２）作用下培养６ｈ的细胞环氧合酶２（ＣＯＸ－２）和血栓素合酶（ＴＸＳ）的ｍＲＮＡ。结果：ＨＩＦ－１诱导剂ＣｏＣｌ２可放ＣＯＸ－２和ＴＸＳ基因转明显增强２,向细胞导入野生ＥＰＯ３＇增强子片断可阻断ＣｏＣｌ２诱     

内皮素-1 mRNA反义寡核苷酸预防大鼠急性缺血性心律失常

本实验夹闭雄性ＳＤ大鼠冠状动脉左前降支（ＬＡＤ）造成急性心肌缺血,观察ＬＡＤ闭塞后１ｈ内缺血性心律失常的发生。在ＬＡＤ夹闭前２ｈ,静脉注射本室设计的人内皮素－１ｍＲＮＡ反义寡核苷酸（ＡＳ－ＯＤＮ）以阻断ＥＴ－１ｍＲＮＡ表达,观察ＡＳ－ＯＤＮ对血浆ＥＴ－１浓度和急性缺血性心律失常的影响。     

The NO donor molsidomine reduces endothelin-1 gene expression in chronic hypoxic rat lungs

We investigated the effects of the nitric oxide (NO) donor molsidomine and the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) on pulmonary endothelin (ET)-1 gene expression and ET-1 plasma levels in chronic hypoxic rats. Two and four weeks of hypoxia (10% O2) significantly increased right ventricular systolic pressure, the medial cross-sectional vascular wall area of the pulmonary arteries, and pulmonary ET-1 mRNA expression (2-fold and 3.2-fold, respectively). ET-1 plasma levels were elevated after 4 wk of hypoxia. In rats exposed to 4 wk of hypoxia, molsidomine (15 mg x kg(-1) x day(-1)) given either from the beginning or after 2 wk of hypoxia significantly reduced pulmonary hypertension, pulmonary vascular remodeling, pulmonary ET-1 gene expression, and ET-1 plasma levels. L-NAME administration (45 mg x kg(-1) x day(-1)) in rats subjected to 2 wk of hypoxia did not modify these parameters. Our findings suggest that in chronic hypoxic rats, exogenously administered NO acts in part by suppressing the formation of ET-1. In contrast, inhibition of endogenous NO production exerts only minor effects on the pulmonary circulation and pulmonary ET-1 synthesis in these animals.     

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

慢性低氧对大鼠肺内几种原癌基因表达的影响

本文用原位杂交技术动态观察了慢性低氧大鼠肺内原癌基因ｊｕｎ、ｆｏｓ和ｍｙｂ的表达,结果发现：（１）正常大鼠肺内有一定量的ｊｕｎｍＲＮＡ表达,少见到ｍｙｂ及ｆｏｓ的表达;（２）低氧１周时,ｊｕｎ的表达较正常下降,２周后有所增加。低氧３周后,ｊｕｎ的ｍＲＮＡ表达较正常明显上升;（３）低氧１、２周后,ｍｙｂ的表达明显增加,３周时基本恢复到正常水平;（４）ｆｏｓ原癌基因在低氧１、２周时有一定量的表达,低氧３周时达最大值。提示低氧可刺激大鼠肺内原癌基因ｊｕｎ、ｍｙｂ和ｆｏｓ的表达。     

Impaired iNOS-sGC-cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS-sGC-cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague-Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight-matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle+septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α(1) mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension.     

SENP1在大鼠低氧性肺动脉高压形成中肺血管壁中表达及可能作用

目的:探讨大鼠低氧性肺动脉高压(HPH)形成过程中SENP1在肺小动脉的动态表达变化及作用。方法:40只成年雄性Wistar大鼠随机分为5组(n=8):对照组和缺氧3 d、7 d、14 d2、1 d组,常压间断低氧复制HPH大鼠模型。测各组大鼠平均肺动脉压(mPAP)、右心室肥大指数(RVHI)、血管形态学指标;原位杂交、逆转录-聚合酶链反应(RT-PCR)检测肺内SUMO特异性蛋白酶-1(SUMO-specific proteases-1,SENP1)mRNA表达,免疫组化、Westernblot检测其蛋白质水平。结果:①缺氧7 d后,肺小动脉出现血管重塑,且mPAP明显上升;低氧14 d后,肺小动脉重塑更明显,mPAP达高峰。RVHI在低氧14 d后明显增加。②原位杂交显示,SENP1 mRNA在对照组肺小动脉壁呈阳性表达,低氧后其相对量无明显变化。RT-PCR显示肺组织SENP1 mRNA表达与原位杂交所观察到的肺小动脉壁SENP1 mRNA变化趋势一致;SENP1蛋白在对照组呈阳性表达,低氧7 d后其表达量开始呈进行性下降。Western blot显示肺组织内SENP1蛋白表达与免疫组化观察到的肺小动脉壁SENP1蛋白变化趋势一致。③SENP1蛋白与mPAP、重塑指数、RVHI均呈负相关。结论:慢性低氧诱导肺小动脉壁SENP1蛋白降解,进而可能在HPH发病过程中发挥一定的作用。     

Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats.

To investigate the effect of pulmonary alveolar hypoxia on the synthesis and release of endothelin (ET)-1, ET-1-like immunoreactivity (-LI) levels of the lung and plasma were measured in conscious unrestrained rats under hypoxic conditions. Sixty-min exposure to alveolar hypoxia (10% O2 or 5% O2) increased the ET-1-LI level in the lung. The plasma ET-1-LI level in hypoxic rats also increased significantly. The increase of plasma and lung ET-1-LI levels were parallel to the severity of hypoxia. These results demonstrates that acute pulmonary alveolar hypoxia increases lung and plasma ET-1-LI levels in conscious unrestrained rats, suggesting a possible physiological or pathophysiological significance of ET in alveolar hypoxia.     

Effects of ET-A receptor blockade on eNOS gene expression in chronic hypoxic rat lungs

We tested the hypothesis that pulmonary endothelial nitric oxide synthase (eNOS) gene expression is primarily regulated by hemodynamic factors and is thus increased in rats with chronic hypoxic pulmonary hypertension. Furthermore, we examined the role of endothelin (ET)-1 in this regulatory process, since ET-1 is able to induce eNOS via activation of the ET-B receptor. Therefore, chronic hypoxic rats (10% O(2)) were treated with the selective ET-A receptor antagonist LU-135252 (50 mg x kg(-1) x day(-1)). Right ventricular systolic pressure and cross-sectional medial vascular wall area of pulmonary arteries rose significantly, and eNOS mRNA levels increased 1.8- and 2.6-fold after 2 and 4 wk of hypoxia, respectively (each P < 0.05). Pulmonary ET-1 mRNA and ET-1 plasma levels increased significantly after 4 wk of hypoxia (each P < 0.05). LU-135252 reduced right ventricular systolic pressure, vascular remodeling, and eNOS gene expression in chronic hypoxic rats (each P < 0.05), whereas ET-1 production was not altered. We conclude that eNOS expression in chronic hypoxic rat lungs is modified predominantly by hemodynamic factors, whereas the ET-B receptor-mediated pathway and hypoxia seem to be less important.     

左旋精氨酸对低氧性肺动脉高压治疗作用的实验研究

目的：探讨结构型一氧化氮合酶（ｃＮＯＳ）,内皮素－１（ＥＴ－１）在低氧性肺动脉高压（ＨＰＨ）发病中的机制及左旋精氢酸（Ｌ－Ａｒｇ）对ＨＰＨ的治疗作用。方法：３０只健康雄性ＳＤ大鼠平均分为三组：正常对照组（ＮＣ组）、低氧组（ＨＰ组）、低氧左旋精氨酸治疗组（ＬＴ组）。后组每日低氧前给予２００ｍｇ／ｋｇ Ｌ－Ａｒｇ。于低氧２１ｄ检测运动血流动力学,肺组织ＮＯ、ＥＴ－１含量,肺动脉内皮ｃＮＯＳ含量的改变,