基因学干预研究：血红素氧合酶1微卫星多态性表达水平上调的作用

背景：人类血红素氧合酶1(Heme Oxygenase-1，HO-1)基因启动子区域有一双核苷酸(GT)n重复序列，有高度多态性，又称为微卫星多态性，体外实验表明通过测定GT重复次数可间接了解人体内HO—1的表达水平。目的：探讨HO—1基因启动子双核苷酸GTn重复序列的微卫星多态性与冠状动脉支架术后再狭窄的关系。设计：以接受冠状动脉支架置入术的冠心病患者为研究对象的病例对照研究。单位：一所大学医院的心内科病房。对象：研究对象为1996-04／2002-05北京大学第一医院心内科病房成功接受冠状动脉支架置入术的冠心病患者，共118例。纳入标准：支架术后3个月以上行冠状动脉造影随访的冠心病患者；排除标准：冠状动脉造影显示原靶病变管腔直径狭窄&;lt;50％和冠状动脉造影随访时间&;lt;3个月的冠心病患者。入选患者年龄(62&;#177;10)岁，男92例，女26例，所有患者均签署知情同意书。根据美国心肺血液协会的标准定义，将患者分为支架内再狭窄组与无再狭窄组，分别为68例和50例。方法：提取患者外周血DNA，经PCR扩增HO—1微卫星序列后采用Spreadex凝胶电泳来进行基因分型。主要观察指标：HO-1基因启动子微卫星基因型频率及其与再狭窄的关系。结果：携带GT重复&;lt;25次等位基因患者的再狭窄率为47．5％，携带两条GT重复均≥25次等位基因患者的再狭窄率为68．4％(P&;lt;0．05)。经多元回归分析校正冠心病危险因素及介入治疗的相关影响因素后．两组患者的再狭窄率差异仍有显著性意义(OR=0．418，95％可信区间0．197～0．887，P&;lt;0．05)。结论：HO-1基因启动子微卫星多态性与再狭窄相关，对冠心病患者冠状动脉支架置入术后的二级预防有十分重要的意义。     

血红素氧合酶-1表达水平与冠心病临床类型的相关性研究

目的探讨血红素氧合酶-1（HO—1）表达水平与冠心病临床类型的关系。方法将105例冠心病患者分为急性心肌梗死组（AMI组，35例）、不稳定型心绞痛组（UAP组，40例）及稳定型心绞痛组（AP组，30例）。采用免疫组化方法对冠心病患者外周血单个核细胞中HO—1的表达进行定位分析，并通过计算机图像系统分析HO—1表达的程度及强度。通过Western blot对HO-1表达水平进行定量分析，比较各组冠心病患者HO—1的表达情况。结果HO-1表达于胞浆，AMI组HO-1表达明显高于UAP组与AP组（均为P〈0．01），UAP组HO—1表达高于AP组（P〈0．05）。结论HO-1表达水平与冠心病严重程度相关。     

有氧运动对自发性高血压大鼠心肌血红素氧合酶活性及血红素氧合酶-1mRNA表达的影响

目的:观察有氧运动对自发性高血压大鼠(SHR)心肌血红素氧合酶(HO)活性及HO-1mRNA表达的影响,探讨适宜的有氧运动对高血压性心肌肥大的防治作用及机制。方法:雄性Wistar大鼠6只为正常对照组(C组),雄性自发性高血压大鼠16只,随机分为高血压组(S组)和高血压运动组(T组)。T组每日进行60min无负重游泳,每周6次,共9周,用RT-PCR方法检测大鼠心脏HO-1mRNA表达。结果:与C组相比,S组心肌HO活性显著降低(P<0.05);与S组相比,T组心肌HO活性显著升高(P<0.05)。与C组相比,各组心肌HO-1mRNA表达差异均有显著性意义,S组心肌HO-1mRNA表达显著升高(P<0.01);与S组相比,T组心肌HO-1mRNA表达也显著升高(P<0.01)。结论:说明长期有规律的运动可以升高SHR心肌HO活性和HO-1mRNA表达。     

丹参素干预体外低温保存大鼠肝脏血红素氧合酶1的表达

背景:研究发现丹参能够降低诱导型一氧化氮合酶mRNA的表达,减少一氧化氮的产生,抑制肿瘤坏死因子、白细胞介素等炎性因子的分泌,具有抗氧化作用.丹参素作为丹参的重要单体成分,是否具有相同的作用? 目的:验证丹参素干预大鼠肝脏血红素氧合酶1的表达,以及对体外肝脏低温保存肝脏的保护.方法:建立大鼠肝脏低温灌注保存模型.分为3组,对照组术前腹腔注射生理盐水,术中用乳酸林格液灌注;实验组术前腹腔注射生理盐水,术中用丹参素+乳酸林格液灌注;抑制剂组:术前腹腔注射锌原朴啉,术中用丹参素+乳酸林格液灌注.保存0,1,3,6h,分别RT-PCR检测血红素氧合酶1mRNA及蛋白表达的情况,检测细胞线粒体钙离子含量及钙离子ATP酶活性,电镜观察各组肝细胞、线粒体形态改变,光镜观察肝细胞、肝小叶形态改变.结果与结论:实验组肝血红素氧合酶1mRNA及蛋白的表达水平明显比其他两组高(P＜0.05).实验组的肝细胞线粒体钙离子含量明显较其他两组低,Ca2+-ATP酶活性较其他两组高.结果提示,丹参素灌注保存液可以诱导大鼠肝脏中血红素氧合酶1的过表达,延长肝脏低温保存时间.     

血红素氧合酶-1在心血管及运动中的作用

近年研究发现，血红素氧合酶-1(heme oxygenase-1．HO-1)及其代谢产物胆绿素、胆红素、一氧化碳(carbon monoxide，CO)和游离铁参与了机体许多生理和病理过程．发挥着重要作用。下面主要对它在心血管及运动中的作用做一综述。     

大鼠急性脊髓损伤后早期血红素氧合酶-1的表达

目的：研究大鼠急性脊髓损伤后血红素氧合酶-1(HO-1)在脊髓表达的变化。方法：参考Nystroem方法建立大鼠脊髓压迫伤模型(T8-9)，对损伤后早期不同时间点应用免疫组化、病理学及图像分析检测HO-1蛋白的表达。应用化学比色法检测不同时间点脊髓中HO活性的变化。结果：正常脊髓组织内存在少量HO-1蛋白的表达，脊髓损伤后4hHO-1表达增强，16h达高峰，之后渐减弱。与正常组、假手术组相比损伤各组HO活性明显增高(P&;lt;0．05)。结论：脊髓压迫损伤后HO-1表达迅速增强，提示HO-1参与了脊髓继发损伤的病理生理过程，其表达可能具有保护作用。     

血红素氧合酶在危重疾病作用中的研究进展

血红素氧合酶(Heme Oxygenase,HO)是血红素分解代谢的退速酶,能催化血红素转化为胆绿素、一氧化碳和游离铁.近年来研究发现,HO-1及其降解产物参与了机体许多生理和病理过程.发挥着重要的保护作用.本文回顾了在不同的动物实验和临床研究上HO-1对组织保护作用的研究进展.而这些都是与危重疾病相关,强调HO-1在组织中诱导而起保护作用,预示着其对危重症的治疗具有重要意义.……     

中药对脑出血后含铁血红素氧合酶-1蛋白表达的影响

目的:研究脑出血后脑内含铁血红素氧合酶-1(HO-1)蛋白的表达以及中药脑溢安的干预作用.方法:用Ⅶ型胶原酶立体定位注入法制备脑出血大鼠模型.100只大鼠随机分为假手术组(20只)、模型组(40只)和脑溢安组(40只,脑溢安4.92 g·kg-1·d-1).各组分别于术后12、24 h及2、4、7 d用免疫荧光双标技术检测脑组织HO1的表达部位和表达细胞种类;用蛋白质免疫印迹法(Western blot)评价HO1蛋白的表达变化.结果:HO-1免疫阳性细胞主要分布在模型组动物患侧基底节,以小胶质细胞为主.Western blot显示:脑出血后HO-1蛋白在12 h即有表达,2 d达高峰,而后逐渐减弱;脑溢安治疗组的表达趋势与模型组相同,2 d时表达水平明显高于以后的时间点.结论:中药脑溢安可促进HO-1的表达,这可能是其通过活血化瘀治疗脑出血、实现脑保护的机制之一.     

丹参对大鼠移植肝血红素氧合酶1表达的影响

背景:器官移植前使用丹参预处理能够保护组织缺血-再灌注损伤,改善移植器官存活率。目的:观察含丹参的冷灌注液对同种异体大鼠移植肝脏中血红素氧合酶1表达的影响,以及对供体肝脏缺血-再灌注损伤的保护作用。方法:将SD雄性大鼠随机分成UW液组(术中使用UW液灌注保存)、丹参+UW液组(术中使用丹参+UW液灌注保存)、ZnPP预处理组(移植前24h腹腔内注射ZnPP,术中使用丹参+UW液灌注保存),建立稳定的大鼠同种异体肝移植模型。同时取10只正常大鼠作为正常对照。结果与结论:丹参+UW液组和UW液组血清总胆红素、谷丙转氨酶、谷草转氨酶水平明显低于ZnPP预处理组(P<0.01)。血红素氧合酶1mRNA及其蛋白在丹参+UW液组中较UW组表达更明显,在ZnPP预处理组中表达明显受到抑制(P<0.05)。丹参+UW液组肝脏Suzuki标准评分明显低于ZnPP预处理组及UW液组(P<0.05)。表明丹参能上调同种异体的大鼠移植肝脏中血红素氧合酶1mRNA及其蛋白的表达,减轻供肝缺血-再灌注损伤,保护移植大鼠肝脏。     

丹参对大鼠移植肝血红素氧合酶1表达的影响

背景：器官移植前使用丹参预处理能够保护组织缺血-再灌注损伤,改善移植器官存活率。目的：观察含丹参的冷灌注液对同种异体大鼠移植肝脏中血红素氧合酶1表达的影响,以及对供体肝脏缺血-再灌注损伤的保护作用。方法：将SD雄性大鼠随机分成UW液组（术中使用UW液灌注保存）、丹参＋UW液组（术中使用丹参＋UW液灌注保存）、ZnPP预处理组（移植前24h腹腔内注射ZnPP,术中使用丹参＋UW液灌注保存）,建立稳定的大鼠同种异体肝移植模型。同时取10只正常大鼠作为正常对照。结果与结论：丹参＋UW液组和UW液组血清总胆红素、谷丙转氨酶、谷草转氨酶水平明显低于ZnPP预处理组（P〈0.01）。血红素氧合酶1mRNA及其蛋白在丹参＋UW液组中较UW组表达更明显,在ZnPP预处理组中表达明显受到抑制（P〈0.05）。丹参＋UW液组肝脏Suzuki标准评分明显低于ZnPP预处理组及UW液组（P〈0.05）。表明丹参能上调同种异体的大鼠移植肝脏中血红素氧合酶1mRNA及其蛋白的表达,减轻供肝缺血-再灌注损伤,保护移植大鼠肝脏。     

慢性阻塞性肺疾病患者血红素加氧酶-1基因启动子多态性对其基因表达的影响

目的:探讨COPD患者血红素加氧酶-1(heme oxygenase-1,HO-1)基因启动子多态性对HO-1基因表达水平的影响.方法:利用PCR、免疫组织化学及原位杂交等技术,在50例肺癌合并COPD组(COPD组)与30例单纯肺癌组(对照组)的肺组织中,检测HO-1基因启动子5'端(GT)n重复序列不同基因型的频率分布特征,及分析其与HO-1基因信使核糖核酸(messenger ribonucleic acid,mRNA)表达及HO-1蛋白表达的关系.结果:①COPD组L/M基因型频率显著高于对照组,而S/S基因型频率显著低于对照组(均为P＜0.05),其它基因型频率比较差异无统计学意义(均为P＞0.05);②HO-1基因启动子(GT)n不同重复序列含有L等位基因样本(Ⅰ组)其HO-1 mRNA和蛋白表达水平显著低于不含有L等位基因的Ⅱ组(均为P＜0.01).结论:HO-1基因启动子5'端(GT)n大量重复序列可能降低了HO-1基因的表达水平,而与COPD的易感性有关.     

Clinical restenosis after coronary stent implantation is associated with the heme oxygenase-1 gene promoter polymorphism and the heme oxygenase-1 +99G/C variant

BACKGROUND: Vascular remodeling after percutaneous coronary stent implantation frequently leads to restenosis. Heme oxygenase 1 (HO-1) is involved in the generation of the endogenous antioxidant bilirubin and carbon monoxide, both of which exert antiinflammatory and antiproliferative effects. The aim of the present study was to evaluate the influence of genetic risk factors combined with the conventional risk factors on the development of coronary restenosis after percutaneous coronary intervention (PCI) with stent implantation. METHODS: The HO-1 gene GT dinucleotide repeat promoter polymorphism and HO-1 +99G/C variant were evaluated in 199 patients with coronary artery disease after coronary stent implantation and control angiography at 6 months after the intervention. Coronary restenosis was confirmed by quantitative angiography. RESULTS: Carriers of the long allele of the HO-1 gene promoter (>29 repeats) had a significantly higher risk of developing restenosis after PCI than noncarriers [odds ratio (OR)=1.9; 95% confidence interval (95% CI), 1.0-3.4; P=0.04]. Interestingly, the allele longer than 29 repeats conferred a significantly higher risk of developing restenosis (OR=3.4; 95% CI, 1.2-9.1; P=0.017) in nonsmokers than in smokers (OR=2.0; 95% CI, 0.7-5.2; P=0.18). CONCLUSIONS: The long allele of the HO-1 gene promoter (>29 repeats) polymorphism, which leads to low HO-1 inducibility, may represent an independent prognostic marker for restenosis after PCI and stent implantation. The effect of the >29 repeat allele is attenuated in smokers, who have chronic exogenous CO exposure.     

血红素氧化酶基因启动子多态性与慢性阻塞性肺疾病及胸部CT表现的关系

目的 探讨中国西南地区汉族人群血红素氧化酶(HOX)-1基因启动子多态性与慢性阻塞性肺疾病(COPD)易感性及胸部CT表现的关系.方法 应用微卫星片段分析法,分析HOX-1基因型在180例COPD吸烟者和150例健康吸烟者中的分布频率;然后对180例COPD患者通过胸部CT检查进行分型;最后分析HOX-1基因型和COPD患者的胸部CT分型之间的关系.结果 根据HOX-1基因启动子(GT)n重复片段大小,将HOX-1等位基因分为三种类型:S等位基因(≤25个GT重复片段)、M等位基因(26～31个GT重复片段)和L等位基因(≥32个GT重复片段);再根据是否含有L等位基因,将HOX-1基因分为两类基因型,Ⅰ类:包含有L等位基因(S/L,M/L,L/L);Ⅱ类:不包含有L等位基因(S/S,S/M,M/M).研究发现:HOX-1基因型中Ⅰ类基因型在COPD吸烟者中的分布显著高于健康吸烟者(29.4%VS 18.7%,P=0.023,OR=1.8,95%CI 1.1～3.1),但是,携带不同HOX-1基因型(Ⅰ类和Ⅱ类)的两组COPD患者,比较其胸部CT表现分型差异无统计学意义.结论HOX-1基因遗传多态性在中国西南地区汉族人群中与COPD的易感性有关,而且,包含有HOX-1 Ⅰ类基因型的个体更易感COPD.但是,HOX-1基因多态性并不会影响COPD患者在胸部CT影像学上的表现分型.     

Induction of long-term cardiac allograft survival by heme oxygenase-1 gene transfer

Elevated expression of heme oxygenase-1 (HO-1), an intracellular enzyme that degrades heme into carbon monoxide (CO), biliverdine and free iron, has anti-inflammatory and antiapoptotic effects in diverse models. Here, we analyzed the effects of specific overexpression of HO-1 following adenovirus-mediated (AdHO-1) gene transfer in an acute cardiac allograft rejection model. The intragraft (i.g.) injection of AdHO-1 into cardiac allografts, as well as intramuscular (i.m.) or intravenous (i.v.) administration, prolonged allograft survival with, respectively, 13.3, 62.5 and 80% of the grafts surviving long term (>100 days), whereas control grafts were rejected with acute kinetics. HO-1 overexpression was associated with inhibited allogeneic responses in MLRs using graft-infiltrating leukocytes and splenocytes, but not with lymph node cells. The inhibition of splenocyte proliferation was mediated by soluble factors and was dependent on the presence of APCs, since purified T cells proliferated normally. i.v. but not i.g. AdHO-1 administration decreased the number of graft-infiltrating leukocytes, cytokine mRNA accumulation and apoptosis in transplanted hearts, whereas i.v. and i.g. AdHO-1 did not modify normal immune responses against cognate antigens, indicating that there was no general immunosuppression. These results indicate that HO-1 overexpression prolongs the survival of vascularized allografts by promoting tolerogenic mechanisms acting on allogeneic cellular immune responses.     

Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain

The R3V6 peptides, which are composed of a 3-arginine block and a 6-valine block, formed self-assembled micelles in aqueous solution. Dye quenching assays showed that a hydrophobic fluorescent dye, 5-dodecanoylaminofluorescein (DAF), interacted with and was loaded into the hydrophobic core of the micelles. In this study, dexamethasone-loaded R3V6 peptide micelles (R3V6-Dexa) were evaluated as a gene carrier. R3V6-Dexa had higher gene delivery efficiency in human embryonic kidney 293 cells compared to those of the R3V6 peptides and poly-L-lysine (PLL). Dexamethasone might stabilize the micelle structure of the R3V6 peptides by forming strong hydrophobic cores and enhanced the transfection efficiency. Furthermore, R3V6-Dexa reduced the expression of an inflammatory cytokine, interleukin-6 (IL-6), more efficiently in lipopolysaccharide (LPS)-induced Raw264.7 cells than did dexamethasone, suggesting that R3V6-Dexa is also a useful carrier for dexamethasone delivery. A focal brain ischemia-reperfusion model was produced by middle cerebral artery occlusion (MCAO). A heme oxygenase-1 (HO-1) expression plasmid DNA, pSV-HO-1, was delivered into the brain using R3V6-Dexa as a carrier. The pSV-HO-1/R3V6-Dexa complex was injected into the brain 1hr prior to MCAO. Twenty-four hours later, the HO-1 expression of the pSV-HO-1/R3V6-Dexa injection group was higher than those of the MCAO control, pβ-Luc/R3V6-Dexa, and pSV-HO-1/PEI25k injection groups. In addition, the infarct size was reduced due to the delivery of pSV-HO-1/R3V6-Dexa complex. Therefore, R3V6-Dexa may be a useful carrier for HO-1 gene delivery and stroke gene therapy.     

Dialysis membrane-induced oxidative stress: role of heme oxygenase-1

Dialysis membranes have been reported to induce monocyte apoptosis. We studied the role of oxidative stress in the induction of dialysis membrane-induced monocyte apoptosis. Superoxide dismutase, a superoxide scavenger, prevented dialysis membrane-induced monocyte apoptosis. Similarly, other antioxidants also inhibited dialysis membrane- induced apoptosis. In addition, the interaction of dialysis membranes with monocytes was associated with the generation of molecules leading to oxidative stress such as superoxide and TBARS. Interestingly, pre-induction of heme oxygenase (HO)-1 by hemin prevented dialysis membrane-induced monocyte apoptosis, whereas inhibition of HO-1 activity (treatment with tin protoporphyrin, SN-P) enhanced dialysis membrane-induced monocyte apoptosis. We suggest that oxidative injury associated with dialysis membrane and monocyte interaction plays a role in monocyte injury. Pre-induction of HO-1 may attenuate dialysis membrane-induced monocyte apoptosis.     

血红素氧化酶1基因重组腺病毒的构建及感染效率鉴定

目的:构建含血红素氧化酶1基因的重组腺病毒及鉴定感染效率。方法:实验于2003-05/2004-06在解放军第三军医大学西南医院消化科实验室完成。①用限制性内切酶XhoⅠ+HindⅢ从克隆载体PRH01中切出血红素氧化酶1基因片段,亚克隆至Puc18中,将之用KpnⅠ+HindⅢ双酶切,再次亚克隆至质粒pAdTrack-巨细胞病毒中,形成转移质粒pAdTrack-Puc18-PRHO1。②将pAdTrack-Puc18-PRHO1PmeⅠ酶切线性化后与腺病毒基因组质粒pAdEasy-1共转化大肠杆菌BJ5183,得到含目的基因的重组体质粒。③重组腺病毒PacⅠ酶切后用脂质体转染293细胞,包装成重组体腺病毒AdH01。④对重组体腺病毒进行鉴定采用聚合酶链反应方法。结果:①利用氯化钙法由pAdTrack-Puc18-PRHO1和pAdEasy-1共转化BJ5183感受态菌,可获得阳性重组体细菌克隆。②重组腺病毒基因组DNA,用目的基因血红素氧化酶1引物进行聚合酶链反应检测,可特异性扩增出356bp的预期条带,而空载体组未能扩增出此片段,表明血红素氧化酶1基因已克隆入重组体腺病毒基因组中,根据绿色荧光蛋白记数法测得腺病毒滴度1.4×1013空斑形成单位/L。③重组腺病毒感染效率的测定结果:用浓缩后的重组腺病毒以感染复数为10感染肾小球内皮细胞,约85%的内皮细胞出现荧光,表明腺病毒AdH01在体外能有效表达相应的基因产物。结论:细菌内同源重组法获得AdH01在体外能有效表达。     

血红素氧化酶1基因重组腺病毒的构建及感染效率鉴定

目的：构建含血红索氧化酶1基因的重组腺病毒及鉴定感染效率。方法：实验于2003—05／2004—06在解放军第三军医大学西南医院消化科实验室完成。①用限制性内切酶XhoⅠ＋Hind Ⅲ从克隆载体PRH01中切出血红素氧化酶1基因片段，亚克隆至Puc18中，将之用KpnⅠ＋HindⅢ双酶切，再次亚克隆至质粒pAdTrack-巨细胞病毒中，形成转移质粒pAdTrack-Pucl8-PRH01。②将pAdTrack-Pucl8-PRHO1 Pme Ⅰ酶切线性化后与腺病毒基因组质粒pAdEasy—1共转化大肠杆菌BJ5183，得到含目的基因的重组体质粒。③重组腺病毒PacⅠ酶切后用脂质体转染293细胞，包装成重组体腺病毒AdH01。④对重组体腺病毒进行鉴定采用聚合酶链反应方法。结果：①利用氯化钙法由pAdTrack-Puc18-PRHO1和pAdEasy-1共转化BJ5183感受态菌，可获得阳性重组体细菌克隆。②重组腺病毒基因组DNA，用目的基因血红素氧化酶1引物进行聚合酶链反应检测，可特异性扩增出356bp的预期条带，而空载体组未能扩增出此片段，表明血红素氧化酶1基因已克隆入重组体腺病毒基因组中，根据绿色荧光蛋白记数法测得腺病毒滴度1．4&;#215;10^13空斑形成单位／L。⑧重组腺病毒感染效率的测定结果：用浓缩后的重组腺病毒以感染复数为10感染肾小球内皮细胞，约85％的内皮细胞出现荧光，表明腺病毒AdH01在体外能有效表达相应的基因产物。结论：细菌内同源重组法获得AdH01在体外能有效表达。