【作者】 周东海；

【导师】 郭定宗； 熊远著； 邓昌彦； 陈焕春； 郑用琏； 喻传洲； 龙良启；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2005， 博士

【摘要】 肉鸡肺动脉高压综合征(pulmonary hypertension syndrome,PHS)亦称肉鸡腹水综合征(ascites syndrome,AS),又称“心衰综合征”(heart failure syndrome,HFS)等,是快速生长肉鸡的一种常见非传染性疾病,以明显的腹水和心、肺功能衰竭为特征。全世界养鸡业每年因此病而损失巨大,随着养鸡业的发展,该病的发生有逐渐上升之势。肺动脉压升高是肉鸡肺动脉高压综合征的重要特点并对它的形成起决定性作用。内皮素(endothelin-1,ET-1)是一种由内皮细胞合成和分泌的、具有强烈收缩血管作用和促进细胞增生的血管活性多肽,心钠素(atrial natriuretic peptide,ANP)、血管紧张素Ⅱ(angiotensin Ⅱ,AⅡ)、NO等在心血管系统功能的调节中也起着重要作用。为研究肉鸡PHS的发病机理,本试验主要从ET-1角度出发,从以下几个方面对本试验进行了研究:用高能日粮饲喂较多肉鸡的方法获得PHS肉鸡模型;用放射免疫分析方法比较了不同腹水程度肉鸡在不同日龄时血浆和主要组织中ET-1的含量,同时还用硝酸还原酶法和放射免疫法对NO、NOS、ANP、AⅡ在肉鸡PHS发病过程中的作用加以研究;通过免疫组织化学染色方法和RT-PCR的方法研究了ETA受体及其基因在不同腹水程度和不同日龄肉鸡体内的分布情况及其表达水平;通过短期静脉注射ET-1、内皮素受体拮抗剂BQ123和长期给予ET-1的方法研究了ET-1、BQ123对肉鸡肺动脉压、PHS发病率、主要组织病理变化以及一些生理生化指标的影响;同时利用细胞培养的方法研究了ET-1、BQ123、L-Arg、L-NAME对培养的肉鸡心肌细胞的作用,从而比较全面地从ET-1的角度研究了肉鸡PHS的发病机理。主要试验结果如下: (1) 用高能日粮饲养肉鸡时从10日龄起有PHS发生,发病率为5.3%-6.7%,与自然情况下的发病率基本相同;右心导管法研究发现2-6周龄时肺动脉高压(PH)组肉鸡的平均肺动脉压(mPAP)极显著(p<0.01)高于正常组肉鸡;腹水心脏指数(ascites heart index,AHI)也极显著(p<0.01)或显著(p<0.05)高于正常组,且随日龄的增长有上升的趋势;PH组肉鸡血浆和主要组织(心、肝、肺、肾)中ET-1在2-5周龄时也显著(p<0.01)或极显著(p<0.05)高于正常组,而在6周龄时则呈现出复杂的变化;血浆ANP、NO、AⅡ在2-5周龄时显著或极显著高于正常组,随日龄的增长有升高的趋势,而在6周龄时则呈现出不规则的变化;几种组织相比,肺脏ET-1的含量最高;mPAP与血浆ET-1相关性显著,与血浆NO没有相关性,血浆ET-1与NO也没有明显的相关性。 (2) 用免疫组织化学染色的方法和RT-PCR的方法对不同腹水程度、不同日龄肉鸡ETAR及其基因在体内定位及其表达变化的情况进行了探讨。免疫组织化学染色更多还原

【Abstract】 Pulmonary hypertension syndrome (PHS) of broilers is also called ascites syndrome in broiler or heart failure syndrome (HFS) in broiler. The term "ascites" actually refers to the fluid accumulated in the abdominal cavity as a consequence of heart failure. A frequent occurrence of ascites has been a difficult problem detrimental to the poultry industry around the world. Approximately 4% of all broilers die from pulmonary hypertension syndrome, causing a loss estimated at $1 billion annually. General agreement exists regarding the reasonable etiology of ascites irresoluble. Now most of the investigators focused on pulmonary hypertension because they thought it was the center link of this disease. Endothelin-1 (ET-1) is a potent vasoconstrictive peptide that was isolated initially from the conditioned medium of cultured endothelial cells. It is a pathogenic mediator with a number of deleterious effects, including vasoconstriction, fibrosis, vascular hypertrophy, and inflammation. Atrial natriuretic peptide, angiotensin Ⅱ and nitric oxide also takes an important role in cardiovascular system. In order to illuminate the etiopathogenesis of pulmonary hypertension syndrome in broiler from the point of endothelin-1, natural pulmonary hypertension syndrome broiler cases were got through using hyperalimentation dietary to feed a great deal of commercial broiler chickens. Radioimmunoassay was used to detect endothelin-1(ET-1), ANP and AⅡ in plasma, heart, liver, lung and kidney of different degree ascites broilers and normal broilers at different weeks of age. The AHI and mPAP of 3 to 6 weeks were measured as well. At the same time, nitrate reductase method was used to detect the level of nitric oxide (NO) and nitric oxide synthase (NOS) in plasma of different degree ascites broilers at the age of 3,4,5 and 6 weeks. Imunohistochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) method were used to illustrate the distribution and changes of ETA receptor in different degree of ascites in broiler. In order to study the effect of ET-1 and BQ123 on the mean pulmonary arterial pressure (mPAP), morbidity of PHS, pathological change of the main tissues, physiological index and biochemical indicator, different dose of ET-1 and BQ123 were i.v. injected into the broiler. Myocardial cells of broiler were also cultured to illustrate the effection of different doses of ET-1, BQ123, L-Arg and L-NAME on the cultured myocardial cells.(1) Natural pulmonary hypertension syndrome broiler cases were appear from the age of 10 days after hyperalimentation dietary were used to feed a great deal of commercial Arbor Acre broiler chickens, the morbility is about 5.3% or 6.7%. Right heart catheterwas used to determine the mean pulmonary arterial pressure, it was found that, compared with the normal group, the AHI and mPAP of PH group were higher or significantly higher, the tendency of increment with the days delaying was observed. From 3 weeks of age, the level of plasma and tissue ET-1 of the PH group was higher or significantly higher than that of the normal group. At 6 weeks of age, the level of ET-1 in plasma, lung, kidney of PH group decreased slightly, though it was still higher than that of the normal group. The level of ET-l of tissues was higher than that of plasma, and the level of lung ET-1 was higher than that of other organs. The level of NO and NOS of the two groups increased with the days delayed, and the PH group was significantly higher than the normal group. Besides, there was a significant positive correlation between mPAP and ET-1, but no significant correlation was found between mPAP and plasma NO of the two groups. It is concluded that ET-1 and NO took part in the formation and development of pulmonary hypertension syndrome in broilers and played an important role in it.(2) Immunohistochemical method and reverse transcriptase polymerase chain reaction (RT-PCR) method were used to study the distribution and expression of ETA receptor (ETAR) in different degree of ascites broilers. It was found that the ETAR expressed extensively in the main tissues of the broilers. In heart, it mainly expressed in cardiac muscle cell envelope, cardiac pericardium and vascular smooth muscle. In liver, it mainly distributed in hepatic cell envelope and Kupffer’s cells. In lung, it mainly expressed in blood vessel, bronchus secundarius, parabronchus and pulmonary alveoli. In distal convoluted tubule, renal glomeruli, macula densa, medullary loop and vessel of kidney of kidney, it was also has different degree of expression. Compared with the normal group, the expression of ETAR in lung blood vessel and pulmonary alveoli of PH group, light ascites group and heavy ascites group is higher or significant higher. But with the development of the degree of ascites, the expression of ETAR was decreased, and the expression of ETAR in the PH group is the highest. With the development of the degree of ascetes, the expression of ETAR of the heart was decreased or significant decreased, but it was still higher than the normal group. The expression of ETAR of myocardium of right ventricle was significant higher than of myocardium of left ventricle. In liver, the expression of ETAR in liver cells was higher than that of the normal group and the heavy ascites group. Compared with the normal group, the expression of ETAR in hepatic blood vessel was obviously decreased. In kidney, the expression of ETAR in nephric tubule, glomeruli of kidney and macula densa also showed complicated changes with the development of the degree of ascites. The primers of broiler endothelin type A receptorgene was used to RT-PCR. The heart cDNA was obtained from the normal group broiler of 16, 30 days old and heavy ascites broiler of 30 days old. The results showed that the expression of EDNRA mRNA of the normal 16days old broilers was obviously lower than that of the 30days old broilers. The expression of EDNRA mRNA of the heavy ascites broiler was obviously lower than that of the normal group of broilers.(3) When different degree of ascites group compared each other, the mean pulmonary arterial pressure, ascites heart index (AHI), right ventricle and body weight ratio (TV/BW), the level of plasma ET-1, plasma ANP and plasma NO were all significantly or every significantly increased with the development of ascites degree. Plasma and tissue endothelin-1 (ET-1), atrial natriuretic polypeptide (ANP), mPAP, AHI and NO of the normal, light and heavy ascites broiler groups were measure. It was found that AHI and mPAP of the light ascites group were higher or significantly higher than that of the normal group, and the heavy ascites group was higher or significantly higher than that of the light ascites group. The trend of increment with the days delaying was observed too. The change tendency of plasma and lung inducible nitric oxide synthase (iNOS), constitutive nitric oxide synthase (cNOS) level was complex. Compared with the normal and light ascites group, the level of plasma iNOS in heavy ascites group were higher or significantly higher. The trend of increment of iNOS with the day delaying was complex, it was decreased at first and then increased. The level of cNOS in heavy ascites was lower or significant lower than the normal and light ascites group. It showed complex change as broilers growing. Radioimmunoassay was used to detect tissue ET-1 and ANP of broilers in different degree ascites group of 5 weeks old broilers. It found that the content of ANP and ET-1 is significantly or very significantly increased with the development of the ascites. The level of the ET-1 and ANP in lung and heart is the highest than in other tissues, and the level of ANP in tissues was higher than that of plasma. Besides, there were significant positive correlation between mPAP and AHI, between plasma ANP, ET-1 and AHI, mPAP. There were significant positive correlation among plasma mPAP, AHI and cardiac ET-1 at 5 weeks of age, but no significant correlation was found between plasma NO and plasma ET-1, plasma ANP, cardiac ET-1.(4) Twenty eight days old broilers were randomly divided into several groups with 8 replicates each, infused intravenously with different dose of ET-1 and BQ123. The control group was injected 0.86% NaCl. The mean pulmonary arterial pressure (mPAP) was measured, and plasma A I , ANP, ET-1 at different time after the broilers were injected different dose of ET-1 and BQ123. The mPAP decreased from time 0 to 5 min and thenbegan to increase from time 5 to 30 min when the broilers were injected with ET-1 24, 120, 240ng/kg BW. The effective dose of ET-1 was 240ng/kg BW to make mPAP increase. BQ123 can decrease mPAP of the broilers with dose dependent and it also can counteract the increasing effect of blood pressure after injected ET-1, but it can’t counteract the decreasing effect of blood pressure at the beginning of the injection. In other experiment, 170 broilers were divided into 3 groups: a control group (group C, 50 broilers), group A and B (60 broilers each) that intravenously injected with 240ng/kg BW and 360ng/kg BW ET-1, respectively. The PHS morbidity of group A was higher than that of group C but lower than group B. The right/total ventricular weight ratio (RV/TV) and mPAP were higher in group A and B than in group C. There was the same tendency of the values of packed cell volume (PCV) and plasma nitric oxide (NO) when compared group A, B with group C. The tissue slice of broilers showed that pathology changes appeared in the main tissues after ET-1 was injected into the broiler with long-term.(5) Enzyme digestion was used to cultivate myocardial cell of broilers. In this experiment, methyl thiazolyl tetrazolium (MTT) and 3H-TdR, 3H-Pro, 3H-Leu were used to study the effect of ET-1 and BQ123 on the broiler myocardial cell. In the present study, it indicated that when the concentration of ET-1 exceeded 10"8mol/L, ET-1 was obviously promoted the growth and hypertrophy of the myocardial cells. If the ET-1 concentration was higher, the growth promotion effect on the myocardial cells was better. When the concentration of BQ123 exceeded 10"8mol/L, it can completely counteract the promotion effect of ET-1 on myocardial cells of broilers, and the inhibitory action was obvious if the concentration of BQ123 was higher. Fluorescent labeling was used to study the effect of ET-1, BQ123, L-Arg and L-NAME on the change of [Ca2+]i in myocardial cells. ET-1 could promote [Ca2+]i of myocardial cells inflowing and BQ123 could counteract this effect soundly, but BQ123 could not counteract the transient increase effect of ET-1.更多还原