【作者】 臧莹安；

【导师】 王小龙；

【作者基本信息】 南京农业大学 ， 兽医， 2006， 博士

【摘要】 肉鸡肺动脉高压综合征（Pulmonary hypertension syndrome，PHS）又称肉鸡腹水综合征（Ascites syndrome of broilers，ASB），是当今世界各国肉鸡业中的一大难题，常常发生在海拔3000米左右的高原地区，主要以肺动脉高压、右心室肥大、心脏衰竭和腹水等症状为特征。近年来，在低于海拔1000-2000米甚至低于海平面的一些地区也常见肉鸡发生类似的疾病。肉鸡PHS是一个复杂的综合征，在美国肉鸡PHS的死亡率为2-5％，甚至高达10％以上，每年因此病造成的经济损失达1亿多美元。在世界范围内的发病率为4.2％，每年约有70亿肉鸡遭受此病的侵害，估计全世界因此病所造成的经济损失每年达10亿美元。从1986年起我国十几个省市也陆续报道了本征，其死亡率为1-30％不等，损失惨重。因此引起了国内外学者对该病的广泛关注。为了攻克这一威胁肉鸡业的世界性难题，国内外许多学者潜心致力于PHS的病因、病理发生和防制的研究。多数学者认为导致肺动脉高压的主要因素是由于在遗传育种中片面追求高的生长速率，其机体代谢加强和对氧的需要量剧增，致使组织代谢性缺氧，引起血液红细胞压积、血液粘度、红细胞脆性和血容量增加等血液流变力学的变化。前人对自由基在肉鸡肺动脉高压综合征的发生、发展过程中的作用及其对自由基的清除作了大量的研究工作，但肺动脉高压综合征肉鸡线粒体内自由基的产生机制如何?线粒体功能有何改变?这些还不十分清楚。阐明其深层次的发病机制，以寻找有效的防治方法具有重要的现实意义。本研究采用黄羽肉鸡120只，随机分为A、B、C三组各40羽，其中A组为对照组，B组为发病组，C组为治疗组。A、B、C组参试鸡14日龄前常规饲养。14日龄后分别采用不同处理：A组鸡仍常规饲养，而B、C两组鸡舍温按每日1～2℃由25℃逐步降至12℃，同时日粮中按1.5 mg·kg^-1的剂量添加T3（sigma公司提供）以诱发肉鸡腹水。另外，自14日龄起，C组在日粮中按500 mg·kg^-1的量添加维生素C进行防治，至试验结束。并于处理后1、2、3、4、5周（即肉鸡3、4、5、6、7周龄时）分别从每组肉鸡中随机抽取6羽进行心、肝、肠粘膜等组织样品的采集，以测定其线粒体中的MDA、SOD、GSH-P_X、NOS、NO水平和Na⁺-K⁺—ATPase的活性，进而探讨肺动脉高压综合征时肉鸡线粒体的抗氧化能力、NO代谢以及评价线粒体氧自由基的产生与各种线粒体电子传递链复合体活性的关系。进一步探讨自由基清除剂对线粒体内自由基生成的影响，为临床用药提供理论依据。1、本实验结果显示，对于发生肺动脉高压综合征的肉鸡，在处理后1周、2周时，其肝脏、心肌和肠粘膜线粒体MDA的含量显著低于正常对照组（P＜0.05），T-SOD活性、GSH-P_X活性却显著提高（P＜0.05）；3周、4周、5周时，其肝脏、心肌和肠粘膜线粒体MDA的含量显著高于正常对照组（P＜0.05）。线粒体T-SOD的活性却显著降低（P＜0.05）。GSH-P_X的活性也显著降低（P＜0.05）。而经治疗后，1周时，发病组的肝脏和肠粘膜线粒体MDA的含量没有能显著增加，心肌线粒体MDA的含量有所增加，但不显著，发病组的肝脏和肠粘膜线粒体T-SOD的活性没有能显著降低，肠粘膜线粒体T-SOD的活性却显著降低；治疗组肝脏线粒体的GSH-P_X活性显著降低，心肌和肠粘膜线粒体的GSH-P_X活性虽然有所降低，但却不显著；2周时，发病组的肝脏和肠粘膜线粒体MDA的含量显著增加，心肌线粒体MDA的含量增加，却不显著。发病组的肝脏和肠粘膜线粒体T-SOD的活性没有能显著降低，肠粘膜线粒体T-SOD的活性却显著降低；2周时，治疗组未能显著降低肝脏、心肌和肠粘膜线粒体GSH-P_X的活性；3周、4周、5周时，肝脏、心肌和肠粘膜线粒体MDA的含量显著降低，且基本降至正常水平（P＜0.05）。T-SOD的活性和GSH-P_X的活性均显著增加（P＜0.05），且最终恢复至对照组水平。结果表明：肉鸡肺动脉高压综合征显著诱导了肉鸡组织细胞和线粒体的脂质过氧化作用，促进了机体自由基生成增多，并显著降低了肉鸡组织细胞和线粒体的抗氧化能力；治疗后，药物能显著抑制肉鸡肺动脉高压综合征诱导的脂质过氧化作用，阻止了自由基的生成，并能显著增强机体的抗氧化能力。2．本实验结果显示，对于发生肺动脉高压综合征的肉鸡，在处理后1周、2周时，其肝脏、心肌和肠粘膜线粒体的NO含量和NOS的活性显著提高（P＜0.05）；3周、4周、5周时，其肝脏、心肌和肠粘膜线粒体NO含量和NOS的活性却显著降低（P＜0.05）。治疗组和发病组相比，1周时，肝脏和肠粘膜线粒体的NO活性显著降低（P＜0.05），但未能降至对照组水平。心肌线粒体的NO活性显著降低（P＜0.05），且基本降至对照组水平；治疗组肝脏线粒体的NOS活性未能显著降低，和对照组间差异显著。心肌和肠粘膜线粒体的NOS活性显著降低（P＜0.05），和对照组间差异显著；2周时，肝脏线粒体的NO活性显著降低（P＜0.05），且基本降至对照组水平。心肌和肠粘膜线粒体的NO活性显著降低，但仍未降至对照组水平；治疗组肝脏线粒体的NOS活性显著降低（P＜0.05），且基本降至对照组水平。心肌和肠粘膜线粒体的NOS活性显著降低（P＜0.05），和对照组间差异显著；3周、4周、5周时，肝脏、心肌和肠粘膜线粒体NO活性和NOS活性均显著增高（P＜0.05），最后基本恢复至对照组水平。结果表明：缺氧的早期，NOS活性升高从而使NO水平升高，NO水平升高舒张血管，缓解肺动脉高压，是机体的一种应激反应。随着缺氧的时间延续，机体对应激因子的反应不能从根本上缓解缺氧，就对此就产生了适应，NOS和NO合成相对不足，相应器官的功能受到损害，肺血管舒缩失衡出现肺动脉高压，进一步发生腹水。药物对此过程具有明显的颉颃作用，对机体起到保护作用。3．本实验研究显示，对于发生肺动脉高压综合征的肉鸡，在处理后1周、2周时，肺动脉高压综合征肉鸡组的肝脏、心肌和肠粘膜线粒体的Na⁺-K⁺—ATPase．活性显著提高，3周、4周、5周时，肺动脉高压综合征肉鸡组的肝脏、心肌和肠粘膜线粒体Na⁺-K⁺—ATPase的活性却显著降低。出现了先激活后顿抑的效应。和发病组相比，1周时，治疗组肝脏线粒体的Na⁺-K⁺—ATPase活性显著降低（P＜0.05），和对照组间差异显著；治疗组心肌、肠粘膜线粒体的Na⁺-K⁺—ATPase．活性未能显著降低，但和对照组间差异显著。2周时，治疗组肝脏线粒体的Na⁺-K⁺—ATPase．活性显著降低（P＜0.05），且基本降至对照组水平；治疗组心肌和肠粘膜线粒体的Na⁺-K⁺—ATPase．活性显著降低（P＜0.05），和对照组间仍存在显著差异。3周、4周、5周时，治疗组肝脏、心肌和肠粘膜线粒体Na⁺-K⁺—ATPase．的活性均显著增高（P＜0.05），且最终达到正常水平。结果表明：肉鸡肺动脉高压综合征诱导了线粒体的损伤，引起了能量代谢障碍，ATP生成减少，线粒体内的体积、渗透压、内环境、静息电位和离子交换发生紊乱，导致血管通透性增加，白细胞和蛋白渗出增加；药物对此过程具有明显的颉颃作用，对机体起到保护作用。4．低温和T₃已成功地诱发了肉鸡PHS，其发病机制可能与肉鸡已产生了大量的自由基并发生了一系列的脂质过氧化链锁反应有关。5．日粮添加维生素C明显阻断了低温和T₃条件下肉鸡体内脂质过氧化过程，有效清除了体内自由基，显著增强了体内抗氧化能力，成功防制了肉鸡PHS的发生。更多还原

【Abstract】 Pulmonary hypertension syndrome （PHS） in broiler chickens poses a serious problem to the international poultry industry, and is an important cause of mortality that has been reported from many parts of the world. For broilers kept at altitudes of approximately 3000m, the onset of pulmonary hypertension, cardiac hypertrophy, heart failure, and oedema leading to PHS has been described. More recently similar symptoms and mortality were reported in broilers reared at lower altitudes of 1000 to 2000m and at altitudes down to sea level. A survey in the United States showed that PHS accounts forover 2-5% of annual broiler losses. An incidence of PHS of 5-10% was reported in China. A global incidence of 4-5% PHS has been reported and, at today’s market prices, this mortality represents a cost to the industry of about one billion＄US annually. It has been suggested that the fast-growing modem broiler is particularly susceptible to PHS, because rapid growth causes an increased demand for oxygen, which forces the heart to maintain an elevated cardiac output. The resulting increase in blood pressure within the pulmonary circulation can cause hypertrophy of the fight ventricle and eventually congestive heart failure. This pathogenesis for PHS includes increased oxygen requirements by body tissues and low oxygen tensions in arterial blood. Though a large amount of research about the function of free radicals during the course of pulmonary hypertension syndrome （PHS） in broiler chickens has been done, but what is the mechanism of an internal free radical of mitochondria? What changes does mitochondria function have? Further pathologic mechanism is not clear. The present study was conducted to further evaluate the pathogenesis of the acute and chronic forms of PHS, and to obtain the methods for preventing PHS.A total of 120 commercial broilers were divided randomly into three groups that were respectively treated as control （Group A） and 2 experimental groups （B、C）, Broilers in different groups were fed a normal diet before 14 days of age. Then In group B and C, broilers were subjected to a change of temperature by lowering 1～2℃per day from 25℃ （day 14） down to 12℃, and maintained at this ambient temperature until 7 weeks of age. At the same time, 1.5 mg·kg^-1 3, 3, 5-triiodothyronine （T₃） in the diet was added to in groups B in order to induce pulmonary hypertension syndrome （PHS）, and 500 mg·kg^-1 ascorbic acid （Vitamin C） in the diet was added to in groups C with the purpose of decreasing the incidence of PHS, Moreover, the liver, heart and mucous membrane of intestine, were taken from 6 killed birds per group at five time points, i. e. after different treatment 1 week,2 weeks,3 weeks,4 weeksand 5 weeks . At every time point, the mitochondria were determined for the evaluations of the concentrations of malondialdehyde （MDA）, nitrix oxide（NO） and the activity of nitric oxide synthase （NOS）, superoxide dismutase （SOD）、glutathione peroxidase （GSH-Px）, Na⁺-K⁺—ATPase.The results indicated that1. At 1 and 2 weeks, the MDA level in PHS group decreased significantly （P＜0.05）, and the activities ofT-SOD and GSH-Px increased markedly （P＜0.05）, of the mitochondria in liver, heart and intestinal mucous membrane. But from 3 weeks to 5 weeks, the MDA level increased significantly, and the activities of T-SOD and GSH-Px decreased markedly （P＜0.05）, of the mitochondria in liver, heart and intestinal mucous membrane.After the treatment of Vitamin C, at 1 week, Vitamin C did not significantly increase the MDA level （P＞0.05）, of the mitochondria in liver, heart and intestinal mucous membrane in broilers with PHS, Vitamin C didn’t significantaly decrease the T-SOD level of the mitochondria in liver and heart （P＞0.05）, but changed the T-SOD level of the mitochondria in intestinal mucous membrane. （P＜0.05）. and the activities of GSH-Px decreased a little, but not markedly （P＞0.05）, of the mitochondria in heart and intestinal mucous membrane, the activities of GSH-Px decreased markedly of the mitochondria in liver （P＜0.05）. At 2 weeks, the MDA level increase significantly （P＜0.05）, of the mitochondria in liver, and intestinal mucous membrane in broilers with PHS, of the mitochondria in heart, this increase is not significantly （P＞0.05）. Vitamin C didn’t significantaly decrease the T-SOD level of the mitochondria in liver and intestinal mucous membrane. （P＞0.05）, but of the mitochondria in heart decreased significantaly （P＜0.05）. and the activities of GSH-Px decreased a little, but not markedly （P＞0.05）, of the mitochondria in liver, heart and intestinal mucous membrane, from 3 weeks to 5 weeks, Vitamin C decreased sugnificantly the activity of MDA, of the mitochondria in liver, heart and intestinal mucous membrane in broilers with PHS （P＜0.05） , the T-SOD and GSH-Px level were increased significantly （P＜0.05）. It might be concluded that the Pulmonary hypertension syndrome （PHS） in broiler chickens induced markedly the lipid peroxidation of mitochondria and cells, and increased clearly the free radicals, and reduced significantly the antioxidative capacity; the Vitamin C significantly stopped lipid peroxidation induced by PHS, and eliminated markedly productions of a lot of free radicals, and increased significantly the antioxidative capacity.2. From 1 week to 2 weeks, the NO and NOS level of the mitochondria in liver, heart and intestinal mucous membrane in broilers increased significantly （P＜0.05）. But from 3 week to 5 week, the NO and NOS level of the mitochondria in liver, heart and intestinal mucous membrane in broilers decreased significantly （P＜0.05）.After the treatment of Vitamin C, at lweek, Vitamin C significantly decreased the NO level （P＞0.05）, of the mitochondria in liver, heart and intestinal mucous membrane in broilers with PHS, but the NOS level of the mitochondria in liver didn’t signifieantaly decrease （P＞0.05）, the NOS level of the mitochondria in heartr and intestinal mucous membrane, significantaly decreased （P＜0.05）. at 2 weeks, Vitamin C significantly decreased the NO level and NOS level, of the mitochondria in liver, heart and intestinal mucous membrane in broilers with PHS（P＜0.05）, From 3 weeks to 5 weeks, the NO level and NOS level, of the mitochondria in liver, heart and intestinal mucous membrane in broilers with PHS were significantly increased.It might be concluded that during the early period of the oxygen deficiting, the activeness of NOS elevates, thus causes the NO level elevate, this diastoles the vessel, alleviates the high pressure of pulmonary artery, is one kind of body stress reaction. But if the oxygen deficiting continuring, body can not react efficiently to alleviate this, thus the adaption comes into being, the synthesize of NOS and NO relatively insufficient, The function of corresponding organ is damaged, the unbalancedly stretch and constringency of the lung blood vessel results the pulmonary artery high pressure, furtherlead to the Pulmonary hypertension syndrome （PHS）. It is obviously that medicine has obviously antagonistic role. This can protect the organism.3. At 1 and 2 weeks, the activity of Na⁺-K⁺—ATPase of the mitochondria in PHS group decreased significantly （P＜0.05） , of the mitochondria in liver, heart and intestinal mucous membrane. But from 3 weeks to 5 weeks, the activity of Na⁺-K⁺—ATPase of the mitochondria in liver, heart and intestinal mucous membrane in broilers decreased significantly （P＜0.05）.Compared with the PHS group, After the treatment of Vitamin C, at 1 week, Vitamin C significantly decreased the the activity of Na⁺-K⁺—ATPase （P＜0.05）, of the mitochondria in liver, but the activity of Na⁺-K⁺—ATPase didn’t significantaly decrease （P＞0.05）, of the mitochondria in heart and intestinal mucous membrane, at 2 week, Vitamin C significantly decreased the activity of Na⁺-K⁺—ATPase, of the mitochondria in liver, heart and intestinal mucous membrane （P＜0.05）, From 3 weeks to 5 weeks, the activity of Na⁺-K⁺—ATPase were significantly increased （P＜0.05）, of the mitochondria in liver, heart and intestinal mucous membrane.It might be concluded that the Pulmonary hypertension syndrome （PHS） in broiler chickens induced the damnification of the mitochondria, resulted the disfunction of the energy metabolism, decreased the production of ATP energy, resulted the impediment of the volume, infiltration press, inner environment, ion exchange, and the increase of the blood vessel penetration and the increase of the effusion of the leucocyte and the albumen. Medicine played an important role in the objection of the pulmonary hypertension syndrome （PHS） in broiler chickens and the protection of broilers.4. The cool ambient and dietary T₃ had successfully induced broiler PHS, which was associated with productions of a lot of free radicals and a series of the chain reaction of lipid peroxidation.5. The dietary vitamin C clearly stop the lipid peroxidation, eliminate the free radicals, increase the antioxidative capacity and reduce the incidence of PHS in broilers in the cold ambient and dietary T₃.更多还原