【作者】 金强；

【导师】 陈卫民；

【作者基本信息】 中国医科大学 ， 麻醉学， 2007， 博士

【摘要】 目的肺动脉高压（PH）是临床上以肺动脉压（MPAP）和／或肺血管阻力（PVR）增高为特点的一组疾病，是多种心肺疾患的常见并发症，亦是心血管外科围手术期难题之一，其严重程度往往决定病人的预后。PH虽致病因素不同、发病机制不清，但具有相似的特点，即肺血管收缩和血管壁重塑（内膜纤维化、中层平滑肌增生肥大、细胞外基质蛋白沉积异常增多），导致了肺血管阻力的增加。研究表明，内皮功能障碍在PH发病机制中起关键作用。正常情况下，肺血管内皮细胞通过内皮素（ET）通路、NO／cGMP通路和前列环素／cAMP通路间的平衡，维持肺循环低张力、高容量状态。此平衡一旦被打破（NO／cGMP、前列环素／cAMP通路功能低下，或ET通路功能上调），即可诱发PH。研究表明，病理情况下NO／cGMP相对不足是PH发病的重要环节。依此理论，NO吸入已成功应用于PH的临床治疗，并取得了可喜的成果。NO吸入虽可高效、选择性地舒张肺血管，改善氧合，并无体循环作用。但价格昂贵、使用不便，并需特殊的吸入装置，且半衰期短、易产生耐药现象和停药后反弹。由于内源性NO是在一氧化氮合酶（NOS）的催化下生成的，同时NOS的三种亚型（神经型NOS（nNOS）、内皮型NOS（eNOS）和诱导型NOS（iNOS））中，eNOS源性NO在维持肺血管张力方面起主要作用。因此，通过基因转移的方法，将外源eNOS基因导入体内，可通过表达产生的eNOS，使内源性NO持续增加，从而对PH起治疗作用，且可避免NO吸入所伴随的不良反应。由于NO通过第二信使cGMP发挥肺血管舒张作用。因此，细胞内cGMP的水平，在PH的治疗中起中心作用。研究表明，细胞内cGMP水平取决于其合成和水解。在体内，cGMP主要通过磷酸二酯酶（PDEs）水解。哺乳动物PDEs包括11个家族，50个亚型。其中PDE5富含于肺脏和阴茎海绵体，是肺内水解cGMP最主要的PDE。研究显示，许多PH动物模型，肺血管PDE5表达和酶活性上调，提示PDE5可能参与PH的病理生理。因此，选择性抑制PDE5可通过降低cGMP的水解而增进NO／cGMP作用，成为PH治疗的另一种途径。鉴于此，我们假设联合应用eNOS基因转移和口服西地那非（高选择性PDE5抑制剂），分别通过增进cGMP的合成，和减少cGMP的代谢，对PH有协同性预防作用，效果应强于二者单独应用，且可减少或避免二者大剂量应用所致的不良反应。由于PH发展的一个重要因素是肺泡内低氧，而低氧亦可导致PH。因而，本研究拟建立大鼠低氧性PH模型，应用携带人类eNOS基因的重组腺病毒AdCMVceNOS基因导入，并联合口服枸橼酸西地那非，通过血流动力学、组织学、尘化学、分子生物学等方法，研究腺病毒介导eNOS基因转移联合西地那非，预防急性低氧性肺血管收缩（HPV）和慢性低氧性肺动脉高压（CHPH）的可行性、疗效及相关毒副作用，为此法用于临床奠定理论基础，为PH治疗开创出更为安全有效的新方法。材料1、实验材料重组腺病毒载体AdCMVceNOS病毒DNA由Dr Robert Gerard惠赠重组腺病毒载体AdCMVLacZ东方肝胆医院惠赠pXCl载体加拿大Microbix Biosystems公司腺病毒E1区转化人胚胎肾细胞株293加拿大Microbix Biosystems公司引物Vt01、Vt02、Vt001和Vt002 Takara公司合成1125-cGMP放射免疫试剂盒上海中医药大学小鼠抗人eNOS单克隆抗体BD公司一氧化氮试剂盒南京建成生物工程研究所免疫组化试剂盒北京中杉生物技术公司弹力纤维染色试剂盒北京中杉生物技术公司ECL试剂盒北京中杉生物技术公司X-gal（5-溴-4-氯-3-吲哚-β-半乳糖苷） Takara公司枸橼酸西地那非辉瑞制药公司10％氮氧混合气沈阳液化空气有限公司100％氮气沈阳液化空气有限公司2、实验设备PCR仪德国BIOMETRA公司N02120MAX21g电子天平NAVIGATOR公司TDL-40B离心机上海安亭科学仪器有限公司721型分光光度计上海精密科学仪器有限公司GC-2016放射免疫计数器科大创新股份有限公司TKR-200C小动物呼吸机江西特力麻醉呼吸设备有限公司3、实验动物Wistar大鼠126只沈阳军区总医院实验动物中心方法1、第一部分（1）AdCMVceNOS病毒DNA用脂质体法体外转染293细胞，且在细胞内部包装。经PCR鉴定后在293细胞株内扩增，继而经CsCl密度梯度离心纯化及TCID50法测定滴度。（2）雄性Wistar大鼠54只，随机分为对照（C）组、Ad-LacZ组和Ad-eNOS组，每组18只。分别应用病毒保存液、5×10⁹pFU／ml AdCMVLacZ和AdCMVceNOS各600μl经气管导管注入大鼠肺脏。3、5和17天后，每组各取6只，测定HR和血压，并行肺组织X-gal染色、eNOS免疫组化染色、HE染色、eNOS Westernblot检测及cGMP和NO含量测定。转染病毒后5天的大鼠，同时行肝、脾、肾脏的组织化学染色。2、第二部分雄性Wistar大鼠36只，随机分为常氧（N）组、低氧（H）组、低氧LacZ（H-LacZ）组、低氧eNOS（H-eNOS）组、低氧西地那非（H-S）组和低氧eNOS-西地那非（H-eNOS-S）组，每组6只。首先经气道转基因（N、H和H-S组应用病毒保存液，H-LacZ组应用5×10⁹PFU／ml AdCMVLacZ 600gl，H-eNOS和H-eNOS-S组应用5×109PFU／ml AdCMVceNOS 600μl经气管导管注入大鼠肺脏）。3天后，N组常氧（吸入空气）30min，其它组大鼠急性低氧（吸入10％氧气）30min。H-S和H-eNOS-S组大鼠在低氧前30min，应用0.3％西地那非25mg／kg灌胃，其它组大鼠灌入等量生理盐水（8.33ml／kg）。低氧结束后，测定大鼠体循环血压（SAP）、HR和MPAP，动脉血气分析，检测肺组织cGMP、NO含量。3、第三部分雄性Wistar大鼠36只，随机分为常氧（N）组、低氧（H）组、低氧LacZ（H-LacZ）组、低氧eNOS（H-eNOS）组、低氧西地那非（H-S）组和低氧eNOS-西地那非（H-eNOS-S）组，每组6只。首先经气道转基因（N、H和H-S组应用病毒保存液，H-LacZ组应用5×10⁹pFU／ml AdCMVLacZ 600μl，H-eNOS和H-eNOS-S组应用5×10⁹PFU／ml AdCMVceNOS 600μl经气管导管注入大鼠肺脏）。之后常氧饲养3天，每日灌胃1次。3天后，N组常氧处理（吸入空气）2周，其它组慢性常压低氧处理（吸入10％氧气，每天8h）2周，并于每日低氧前30min灌胃。H-S和H-eNOS-S组应用的灌胃溶液含0.3％枸橼酸西地那非25mg／kg，其余各组灌入等容量生理盐水（8.33ml／kg）。低氧结束后，行血动力学检测、肺血管结构重塑指标检测、右室肥大指标检测、Hct检测，并行肺组织eNOS Westernblot检测及cGMP和NO含量测定。结果1、第一部分（1）AdCMVceNOS病毒DNA经脂质体法成功导入293细胞内，并包装成完整的病毒颗粒。经PCR鉴定并确认，扩增后检测滴度为1.58×10¹⁰PFU／ml。（2）Ad-eNOS组各检测时间点肺组织eNOS蛋白、cGMP和NO含量均明显高于C和Ad-LacZ组（p＜0.01）。免疫组化显示，eNOS转基因表达分布于支气管上皮、肺泡细胞以及肺中、小血管内皮。C和Ad-LacZ组各检测时间点，肺组织eNOS蛋白、cGMP和NO含量无统计学差异（p＞0.05），仅在肺内大血管内皮有内源性eNOS蛋白表达；（3）经气道注入重组腺病毒5天后，Ad-LacZ组大鼠肝、脾、肾脏未见外源LacZ基因表达。但有一只肺组织出现广泛炎细胞浸润。（4）转基因后同一检测时间点，三组大鼠体重（WT）、HR和SAP无明显差异（p＞0.05）。2、第二部分（1）大鼠急性低氧30min后，H-eNOS和H-S组MPAP明显低于H和H-LacZ组（p＜0.01），明显高于H-eNOS-S组和N组（p＜0.01），H-eNOS-S和N组间无统计学差异（p＞0.05）。（2）各低氧组SAP、PaO₂和PaCO₂明显低于N组（p＜0.05），HR明显高于N组（p＜0.05），且各低氧组间无统计学差异（p＞0.05）。（3）N组大鼠肺组织NO含量，明显高于H、H-LacZ和H-S组，明显低于H-eNOS和H-eNOS-S组（p＜0.05），且H、H-LacZ和H-S组间以及H-eNOS和H-eNOS-S组间无统计学差异（p＞0.05）。（4）H和H-LacZ组大鼠肺组织cGMP含量明显低于N组（p＜0.05），H-eNOS和H-S组明显高于N组（p＜0.05），且明显低于H-eNOS-S组（p＜0.01）。3、第三部分（1）各低氧组大鼠体重明显低于N组（p＜0.01），肺脏重量／体重比明显高于N组（p＜0.05）。所有大鼠HR、SAP、肝脏重量／体重比和肾脏重量／体重比无统计学差异（p＞0.05）。（2）低氧各组大鼠MPAP、肺小血管肌化指标（CMA／MA）和右室肥厚指标（RV／（LV+S））均明显高于N组（p＜0.01），H-eNOS与H-S组明显低于H与H-LacZ组（p＜0.01），明显高于H-eNOS-S组（p＜0.05）。H与H-LacZ组间及H-eNOS与H-S组间无统计学差异（p＞0.05）．（3）H-eNOS、H-S和H-eNOS-S组Hct明显高于N组（p＜0.05），明显低于H和H-LacZ组（p＜0.05）。（4）H、H-LacZ和H-S组大鼠，肺组织eNOS蛋白含量明显高于N组（p＜0.01），明显低于H-eNOS和H-eNOS-S组（p＜0.01）。（5）N组大鼠，肺组织NO含量明显高于H、H-LacZ和H-S组（p＜0.05），明显低于H-eNOS和H-eNOS-S组（p＜0.01）。（6）H-eNOS-S组大鼠，肺组织cGMP含量明显高于其它各组（p＜0.01），N、H和H-LacZ组大鼠无明显差别（p＞0.05），明显低于H-eNOS和H-S组（p＜0.01）。结论1、重组腺病毒可介导eNOS基因在大鼠气道高效表达，转基因表达分布广泛（于支气管上皮和肺泡细胞，肺内中、小血管内皮亦有转基因表达），靶向性高，并至少持续17天。外源eNOS基因的表达，可使肺组织NO和cGMP产量明显增高。2、经气道转移重组腺病毒，对大鼠生命体征无影响，但有引发机体免疫反应的可能，故应在达到疗效的同时，尽量控制病毒转移的量或滴度。3、急性低氧30min，可降低大鼠SAP，提高HR，并引发HPV，此病理过程与低氧引起肺组织NO／cGMP含量降低有关。4、慢性低氧二周，可导致大鼠MPAP升高、肺血管结构重塑、右室肥厚和Hct上升，伴肺组织内eNOS表达上调、NO含量降低，而cGMP浓度基本不变。5、经气道吸入AdCMVceNOS和口服西地那非，可增加肺组织内cGMP含量，减轻大鼠急性HPV和慢性低氧引起的MPAP升高、肺血管结构重塑、右室肥厚及Hct升高。二者联合应用，具有协同作用，但不能完全阻止CHPH的病理过程。更多还原

【Abstract】 ObjectivePulmonary hypertension （PH） is a variety of disease states characterized by increased pulmonary artery pressure and/or pulmonary vascular resistance. It is a common complication of the heart and lung disease and also a quiz during cardiovascular surgery perioperative period. The severity of PH always determines the prognosis of patients. Although the pathogenetic factors differs, they all lead to pulmonary vasoconstriction and structural remodeling （e.g. intimal fibrosis, medial hypertrophy, adventitial proliferation, extracellular matrix augment） and ultimately increase PVR. It has been demonstrated, endothelial dysfunction plays a key role in the pathogenesis of PH. Under the normal condition, pulmonary vascular endothelial cells make the pulmonary circulation low resistance and high capacity state by keeping the balance among endothelin （ET） pathway, NO/cGMP pathway and prostacycline/cGMP pathway. The break of the balance leads to the pathogensis of PH.It has been demonstrated, the relative deficiency of NO/cGMP under the patho-condition is the key pathogenetic element of PH. Accordingly, NO inhalation has been successfully used for the treatment of PH, and has made satisfactory results. Though NO inhalation can selectively and efficiently relax pulmonary vessels, improve the oxygenation with no systematic side-effect. It is expensive, unconvenient to be used for requiring a fairly sophisticated delivery system. Furthermore, it has short half-life and can easily produce drug resistance and rebound PH after it’s withdraw. As NO is produced by the catalysis of nitric oxide synthase（NOS）, and among the three submits of NOS, which comprise nNOS, eNOS and iNOS, eNOS derived NO plays the key role in maintaining the pulmonary resistance. So, eNOS can be produced by eNOS gene transfer to elevate endogenous NO, which can treat PH without side-effect accompanied NO inhalation.As NO relax pulmonary vessels through the second messenger cGMP. Intracellular cGMP level plays the key role in treating PH. It has been showed, the intracellular cGMP level depends on the balance between it’s production and degradation. cGMP is hydrolyzed by phosphodiesterases （PDEs）. In mammals, PDEs comprises 11 families and 50 submits. PDE5, which is the main PDE in lungs, abounds in lung and corpus cavemousum. In PH animal models, PDE5 expression and activity in pulmonary vessels upregulates, which indicate PDE5 may participate in the pathogensis of PH. So, selective inhibiton of PDE5 can act by slowing down the degradation of cGMP and hence augment the role of NO/cGMP, which provide a novel strategy in treating PH.So, we hypothesis that a combination of eNOS gene transfer and oral sildenafil（ a highly selective PDE5 inhibitor） can produce a synergistic effect in preventing PH by promoting the production of cGMP and decreasing its degradation. The effect is more powerful than anyone used alone and can avoid the side-effect accompanied at the same time.As one of the factors among the progression of PH is hypoxia in alveoli, and hypoxia can induce PH. So, in our experiment, we establish PH model in rats, and transfer eNOS gene through recombinant adenovirus AdCMVceNOS, combined oral sildenafil. Through hemodynamic, histological, biochemical and molecular biological measures, we investigate the feasibility, therapeutic effect and accompanied side-effect of preventing acute hypoxic pulmonary vasoconstriction（HPV） and chronic hypoxia induced pulmonary hypertension（CHPH） by a combination of eNOS gene transfer and sildenafil. Providing a novel strategy in treating PH.Materials1. Experiment materialsAdCMVceNOS DNA （Dr Rober Gerard）, 293cells and pXC1 vector （Canada Microbix Biosystems company）, primier （Takara company）, 1125-cGMP RIA kit （Shanghai traditional medicine university）, eNOS monoclone antibody （BD company）, NO kit （Nanjing jiancheng biocompany）, immunohistochemical kit and elastic fiber staining kit （Beijing zhongshan biotech company）, Sildenafil citrate （Pfizer company）.2. Experiment instrumentsPCR instrument （Germany Biometra company）, 721 spectrophotometer（Shanghai jingmi scientific instrument limited company）, Animal ventilator（Jiangxi teli anesthesia respiratory instrument limited company）.3. Experiment animals126 Male Wistar rats, provided by Experiment Animal Center, General Hospital of Shenyang Military Area.Methods1. Part 1（1）DNA of virus AdCMVceNOS was transferred into 293 cells by liposome, and packaged inside. After being confirmed by PCR analysis, the virus was amplified in 293 cells and purified by discontinous CsCl gradient. Viral titer was determined with TCID50 method.（2）54 Male Wistar rats were randomized to control（C）group, Ad-LacZ group and Ad-eNOS group（n=18 each）, then Rats were given 600μl of virus conservation solution, AdCMVLacZ or AdCMVceNOS with titer of 5×10⁹pfu/ml by intratracheal instillation respectively. HR and SAP of 6 rats from each group were masured 3, 5 and 17 days after adenovirus transfection. After that, X-gal staining, eNOS immunohistochemical staining, HE staining, eNOS Westernblot of rat lung were performed and cGMP, NO content were measured. X-gal staining of rat liver, spleen and renal were also performed 5 days after virus infection.2. Part 2Thirty six male Wistar rats were randomized to normoxia（N） group, hypoxia（H） group, hypoxia LacZ（H-LacZ） group, hypoxia eNOS（H-eNOS） group, hypoxia sildenafil（H-S） group, and hypoxia eNOS sildenafil（H-eNOS-S） group（n=6）. Gene transfer via airway were performed（N, H and H-S group received virus conservation solution, H-LacZ group received 5×10⁹PFU/ml AdCMVLacZ 600μl, H-eNOS and H-eNOS-S group received 5×10⁹PFU/ml AdCMVceNOS 600μl） on the 1^st day. Three days after adenovirus administration, rats from N group were ventilated with room air for 30min, and rats from other group with 10%O₂. 30min before hypoxia, rats from H-S and H-eNOS-S group were gavaged with 25mg/kg of 0.3% sildenafil. Other rats were gavaged with 8.33ml/kg of NS. SAP, HR and MPAP were measured at the end of hypoxia, arterial blood gas analysis were performed, cGMP and NO content in rat lung were measured.3. Part 3Thirty six male Wistar rats were randomized to normoxia（N） group, hypoxia（H） group, hypoxia LacZ（H-LacZ） group, hypoxia eNOS（H-eNOS） group, hypoxia sildenafil（H-S） group, and hypoxia eNOS sildenafil（H-eNOS-S） group（n=6）. Gene transfer via airway were performed（N, H and H-S group received virus conservation solution, H-LacZ group received 5×10⁹PFU/ml AdCMVLacZ 600μl, H-eNOS and H-eNOS-S group received 5×10⁹PFU/ml AdCMVceNOS 600μl） on the 1^st day. After that, rats were raised three days in room air, gavaged once a day. From the 4^th day, rats apart from N group began hypoxia（10% O₂） exposure 8 hours a day for 2 weeks. Rats from N group were kept in the same room adjacent to hypobaric chamber. Rats were gavaged 30 minutes before hypoxia（normoxia） exposure, H-S and H-eNOS-S group with 0.3%sildenafil 25mg/kg, other groups with 8.33ml/kg of NS. At the end of hypoxia exposure, hemodynamics measurement, Hct, cGMP and NO level in rats lung, pulmonary vascular remodeling index, RV/（LV+S）, and eNOS Westemblot of rats lung were made.Results1. Part 1 （1）DNA of virus AdCMVceNOS was transferred into 293 cells and integrated virus was made. Ater being confirmed and amplified, the virus was quantified to be 1.58×10¹⁰PFU/ml.（2）At each time point, the eNOS protein, cGMP and NO content of Ad-eNOS rats lung were higher than those of C and Ad-LacZ rats （p<0.01）. Immunohistochemical staining showed diffused transgenic expression in bronchi epithelial cells , alveoli lining cells, and endothelial cells in medium- and small-sized pulmonary vessels. In C and Ad-LacZ rats, eNOS, cGMP and NO content of rats lung were similar（p>0.05） and transgenic expression were only observed in endothelial cells in large-sized pulmonary vessels.（3）Five days after adenovirus infection, liver, spleen and renal of Ad-LacZ rats showed no transgenic expression as demonstrated by X-gal staining. However, one from six rats showed diffused inflammation in lung.（4）At each time point, there were no difference in rats weight, heart rate and systematic arterial pressure among 3 group rats（p>0.05）.2. Part 2（1）MPAP of H-eNOS and H-S rats were lower than those of H and H-LacZ rats, higher than those of H-eNOS-S and N rats（p<0.01）. There was no difference in MPAP between H-eNOS-S and N rats（p>0.05）.（2）SAP, PaO₂, PaCO₂ of N rats were higher and heart rate was lower than other rats（p<0.05）. There was no difference among each hypoxia group（p>0.05）.（3）NO content of N rats lung was higher than that of H, H-LacZ and H-S rats, lower than that of H-eNOS and H-eNOS-S rats（p<0.05）. There was no difference among H, H-lacZ and H-S group, and also between H-eNOS and H-eNOS-S group（p> 0.05）.（4）cGMP content of H and H-LacZ rats lung was lower than that of N rats（p< 0.05）. cGMP of H-eNOS and H-S rats was higher than that of N rats（p<0.05）, lower than that of H-eNOS-S rats（p<0.01）. 3. Part 3（1）Weight of N rats was higher and lung weight/body weight ratio was lower than those of other rats（p<0.01）. HR, SAP, liver weight/body weight ratio and renal weight/body weight ratio were similar among all rats（p>0.05）.（2）MPAP, CMA/MA and RV/（LV+S） of N rats were lower than those of other rats（p<0.01）, with H-eNOS and H-S rats lower than H and H-LacZ rats（p<0.01）, higher than H-eNOS-S rats（p<0.01）. There was no difference between H, H-LacZ group and also between H-eNOS, H-S group（p>0.05）.（3）Hct of H-eNOS, H-S and H-eNOS-S rats was higher than that of N rats（p< 0.05）, lower than that of H and H-LacZ rats（p<0.05）.（4）eNOS level of H, H-LacZ and H-S rats lung was higher than that of N rats（p< 0.01）, lower than that of H-eNOS and H-eNOS-S rats（p<0.01）.（5）NO level of N rats lung was higher than that of H, H-LacZ and H-S rats（p< 0.05）, lower than that of H-eNOS and H-eNOS-S rats（p<0.01）.（6）cGMP level of H-eNOS-S rats lung was higher than that of other rats（p<0.01）, with N, H and H-LacZ group lower than H-eNOS and H-S group（p<0.01）. cGMP of N, H and H-LacZ rats lung was similar（p>0.05）.Conclusions1. Recombinant adenovirus can mediate eNOS gene express efficiently in rat airway. Transgenic expression diffused widely （including bronchi epithelial cells , alveoli lining cells, and endothelial cells in medium- and small-sized pulmonary vessels） without distant organ expression. Transgenic expression can last at least 17 days. The expression of eNOS gene upregulate NO and cGMP level in rats lung.2. Transfer recombinant adenovirus via airway showed no interference to rats vital sign, but do has the possibility of inducing immunoreactivity. So the dose and titer of virus used should be limited while reaching the curative effect.3. Thirty minutes exposure to hypoxia can lower SAP, elevate HR of rats, and induce HPV. The pathogenesis is related to downregulation of NO/cGMP level in rats lung.4. Chronic hypoxia exposure can elevate MPAP and Hct, induce pulmonary vascular remodeling, right ventricular hypertrophy, which accompanied with the upregulation of eNOS, NO and cGMP level in rats lung.5. Transfect with AdCMVceNOS and oral sildenafil, can increase cGMP level of lung, reduce rats acute HPV, reduce the elevation of MPAP, Hct, pulmonary vascular remodeling, right ventricular hypertrophy induced by chronic hypoxia. A combination of the two measures can act synergisticly, but cannot utterly prohibit the pathogenesis of CHPH.更多还原