【作者】 谭勋；

【导师】 王小龙；

【作者基本信息】 南京农业大学 ， 临床兽医学， 2004， 博士

【副题名】肺动脉高压肉鸡肺动脉PKCα、NOS和细胞凋亡的变化

【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，AS)，是发生于快速生长的商品代肉鸡的一种营养代谢性疾病。全世界每年约有4％的肉鸡死于PHS，造成高达10亿美元的经济损失：我国PHS发病率平均为4.5％，每年造成的经济损失约为4.68亿美元。在多数情况下，快速生长和环境低温是引起商品肉鸡发生PHS的主要原因，这两种因素均能造成机体代谢需氧增加，并促使心输出量增多。当PHS易感鸡相对缺乏弹性的肺血管不能容纳快速增多的心输出量，或肺血管病理性收缩造成血液通过肺血管的阻力升高时，则导致肺动脉压升高。持续的肺动脉高压引起机体发生一系列病理生理改变，包括全身性组织低氧、肺血管重构、右心衰竭、肝门脉高压和腹水，最终形成PHS，但其发生机制尚不完全明了。本研究从肺动脉高压肉鸡肺动脉蛋白激酶Cα(PKCα)、一氧化氮合酶(NOS)活性及细胞凋亡等变化入手，进一步研究PHS的发病机制，并寻求不同的防治手段和方法。 试验Ⅰ 蛋白激酶Ca(PKCα)与肺血管重构。采用环境低温复制肉鸡PHS模型，观察肺动脉高压肉鸡肺动脉蛋白激酶Cα(PKCα)的表达及其与肺血管重构的关系。160羽艾维茵-2000商品代肉鸡，按常规方法育雏，于14d时随机分为常温对照组(NT组)和低温组(LT组)，每组各80羽。自14d起，LT组舍内温度从28℃以每天1～2℃的速度下降，至21d降至14～12℃，并维持到试验结束。NT组仍继续按常规饲养(21d起舍温控制在20℃)。记录肺动脉高压综合征(PHS)发病率，并分别于24、32、39和45d从各组随机抽样，测定右心室／全心室(RV／TV)、红细胞压积(PCV)和血红蛋白(Hb)；采用计算机病理图像分析手段，检测肺动脉管壁面积／管总面积(WA／TA)、平均中膜厚度(mMTPA)；采用免疫组化方法结合微机图像分析技术，检测肺小动脉光密度值(OD)，以OD值代表PKCα的表达。结果表明：LT组肉鸡PHS发病率较NT组显著升高(P＜0.05)，RV／TV值在45d时升高(P＜0.05)，PCV和Hb值在32d后升高(P＜0.05)，mMTPA值和WA／TA值显著升高(P＜0.05)，PKCα表达增强，且PKCα表达与mMTPA值和WA／TA之间呈显著正相关。博士学位论文:肺动脉高压肉鸡肺动脉PKCa、NOS和细胞凋亡变化的研究 试验nL一精氨酸(L一Arg)对肺动脉一氧化氮合酶(NOS)活性的影响.240羽艾维菌一2000商品代肉鸡，按常规方法育雏，于14d时随机分为常温对照组(NT组)，低温组(LT组)和低温加L一Arg组(LA组)，每组各80羽.自14d起，LT和LA组舍内温度从28℃以每天l一2℃的速度下降，至Zld降至14一12℃，并维持到试验结束.NT组仍继续按常规饲养(Zld起舍温控制在20℃)。此外，LA组自14d起在饲料中按109·kg一，剂量添加L一Arg，直至试验结束.记录肺动脉高压综合征(PHS)发病率，并分别于24、32、39和45d从各组随机抽样，测定右心室/全心室(RV/TV)和血浆一氧化氮(NO)水平.肺组织切片经卜NADPH一d组织化学染色后，采用病理图像分析软件检测直径在100一200拼m的肺小动脉光密度(oD)值，以OD值代表肺动脉NOS活性.结果显示:LT组肉鸡PHS发病率较NT组显著升高(P<0.05)，LA组与NT组相比无显著差异(尸>0.05)，但显著低于LT组(P<0.05);LT组肉鸡RV/TV在45d时较NT组显著升高(P<0.05)，而LA组肉鸡RV汀v值与NT组和LT组相比均无显著差异(尸>0.05);LT组肉鸡血浆NO浓度总体上与NT组差异不显著(尸>0.05)，仅在39d时降低(P<0.05)，而LA组血装NO浓度自32d时起较NT和LT组显著升高(P<0.05);LT组肉鸡肺动脉OD值显著低于NT组(P<0.05)，而LA组肺动脉OD值在32和39d时较LT组显著升高(P<0.05)，并自32d时起达到NT组水平(P>0 .05)0 试验mL一精氛酸(L一Arg)对肺血管重构的影响.240羽艾维菌一2000商品代肉鸡，按常规方法育雏，于14d时随机分为常温对照组(NT组)，低温组(LT组)和低温加L一Arg组(LA)，每组各80羽。自14d起，LT和LA组舍内温度从28℃以每天1一2℃的速度下降，至Zld降至14一12℃，并维持到试验结束.NT组仍继续按常规饲养(Zld起舍温控制在20℃).此外，LA组自14d起在饲料中按109·kg一，剂量添加L一Arg，直至试验结束.记录肺动脉高压综合征(PHS)发病率，并分别于24、32、39和45d从各组随机抽样，测定右心室/全心室(RV厅V)和血浆一氧化氮(NO)水平。采用计算机病理图像分析软件测定肺动脉管壁面积/管总面积认叭/TA和平均中膜厚度(mMT队)，并用原位缺口末端标记法(T uNEL法)对肺动脉凋亡平滑肌细胞进行染色和计数，计算凋亡细胞百分率;采用免疫组化方法和病理图像分析软件，检测肺小动脉光密度值(OD)，以oD值代表蛋白激酶ca(PKca)的表达。结果:LT组肉鸡PHS发病率升高(P<0.05)，其Rv/Tv值在45d时升高(P<0.05)，而LA组PHS发病率和RV汀V值与NT组相比均无显著差异(尸>0.05);LT组肉鸡肺血管mMTPA值和场叭/TA值较NT组升高(P<0.05)，而LA组mMT队与NT组相比差异不显著(P>O刃5)，其场叭/TA值仅在45d时升高(P<0.05); LA组mMTPA值和场叭八人值与摘要LT组相比呈下降趋势，并在32d时显著降低(P<0.05);LT组血浆NO浓度总体上与NT组差异不显著(尸>0.05)，仅在39d时降低(P<0.05); LA组血装NO浓度?更多还原

【Abstract】 Broiler pulmonary hypertension syndrome (PHS), also known as ascites syndrome (AS), is a devastating metabolic disease that affects young, fast-growing commercial broilers, and has been reported from many parts of the world. Worldwide, approximately 4% of all broilers die from PHS, amounting to a loss estimated at $1 billion annually. Only in China, an average incidence of 4.5% was reported, which counts for a loss of about $468 million annually. The primary triggers for PHS under most conditions of commercial broilers growthout include fast growth and cool temperature. Both triggers increase the metabolic requirement for oxygen and thereby force the heart to increase its cardiac output. Pulmonary hypertension is initiated whenever the relatively inelastic pulmonary vasculatrue of susceptible broilers cannot accommodate the rapidly increasing cardiac output at a low pulmonary arterial pressure, or when inappropriate constriction of the pulmonary vasculature directly evaluate the pulmonary vascular resistance to pulmonary blood flow. After the onset of pulmonary hypertension, susceptible broilers proceed through a cascade of events to terminal PHS, including systemic hypoxemia, pulmonary vascular remodeling, right-sided congestive heart failure, increased intravascular pressure in the liver portal system and, ascites. However, the mechanisms for PHS are not well understood. The present study was conducted to evaluate changes of protein kinase Ca (PKCa), nitric oxide synthase (NOS) and apoptosis of smooth muscle cells in pulmonary arterioles during the pathological progression leading to pulmonary hypertension as for further investigation of the pathogenesis of PHS, and to assess different methods preventing PHS.Test 1 The association between activation of PKCa and pulmonary vascular remodeling. One hundred and sixty Avian-2000 commercial broilers were divided equally into a normal temperature control group (NT) and a low temperature group (LL). All thebirds were reared according to normal temperature schedule before day 14, and then, birds in the group LL were exposed to low temperatures by lowing 1-2 per day from 28 (day 14) down to 14-12(day 21), and remained constant until the end of the experiment, while the controls were still reared at normal temperatures. PHS incidence was recorded in each group. Ratios of right ventricle to total ventricle (RV/TV), packed cell volume (PCV) and hemoglobin (Hb) were measured on days 24, 32, 39 and 45, respectively. Vessel wall area to vessel total area (WA/TA), mean media thickness in pulmonary arterioles (mMTPA) were determined by computer-image analytic technique, and the expression of protein kinase Ca (PKCa) in pulmonary arterioles detected by imrnunohistochemical and computer-image analytic technique. PHS incidence amounted to 3.75% of the group NT and 12.5% of the group LT, respectively, and the difference was very pronounced (P < 0.05). Broilers exposed to the cold had elevated RV/TV ratio, PCV, Hb as well as PKCa expression. Cold exposure caused pulmonary vascular remodeling characterized by thickening of media layer and narrowing of the vessel lumen of arterioles. PKCa expression was positively correlated with both the mMTPA and WA/TA.Test 2. The effect of L-arginine on NOS activity. Two hundred and forty Avian-2000 commercial broilers were divided equally into a normal temperature control group (NT), a low temperature group (LT) and a low temperature with supplemental L-arginine group (LA). All birds were brooded at normal temperatures before day 14. Birds in the group LA and LT were subjected to cold temperatures by lowing 1-2 per day from 28 (day 14) down to 14-12 (day 21), and then remained constant until day 49; while birds in the group NT were still reared in normal temperatures. All the birds were fed pelleted diets whereas those kept in the group LA fed the same diets supplemented with 10g kg L-arginine from day 14 to 49. Birds died from PHS were recorded daily and accumulative PHS incidences calculated in the end of the experiment. Ratios of RV/TV, concentrat更多还原