肥大细胞及其主要介质在肉鸡肺动脉高压综合征中的作用研究
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摘要
肺动脉高压(包括肺血管收缩和肺血管重塑)及右心肥大和衰竭是肉鸡肺动脉高压综合征(PHS)发生发展的重要环节。肺循环受神经、局部体液因子调节，其中肺血管自身调节具有重要的作用。前人对肺血管局部的研究多集中在血管内皮细胞，但近年来，在人类和啮齿类动物低氧性肺动脉高压中初步证实，位于血管周围的肥大细胞(MC)在肺血管收缩和肺血管重塑中也具有重要的意义，但MC在肉鸡PHS中的研究未见报道。本试验拟对MC及其主要颗粒介质在PHS发生发展过程中的作用进行探讨，旨在丰富MC生物学资料及PHS发病机理。为此进行如下试验：
1．以环境低温诱发肉鸡PHS，测定各组肺动脉压、股动脉压、右心室压、右心室内压最大变化速率等指标的变化，并对肺血管重塑进行观察，同时对肺实质、肺血管壁、肺血管腔、支气管粘膜、心脏和肝脏中的MC数、MC面积、灰度值进行研究。结果显示，(1)环境低温诱发了肉鸡肺动脉高压综合征，低温组形成肺动脉高压(低温组平均肺动脉压2.47±0.40～3.59±0.47kPa vs常温组1.07±0.02～2.76±0.59kPa)，右心肥大(AHI高达32.53±4.78％)，腹水发生率为14.28％。(2)低温组在肺动脉高压形成、右心肥大及衰竭过程中，肺实质(17.49±1.55％～35.14±3.66％vs9.91±0.87％～18.61±1.76％)、肺血管壁MC脱颗粒率(12.32±1.65％～21.77±3.68％vs 35.28±2.90％～44.77±2.78％)显著(p＜0.05)升高，肺实质(115.44±16.24％～128.48±11.19％vs 72.89±12.25％～83.33±11.85％)、肺血管壁(115.68±12.97％～129.97±14.01％vs 67.15±10.62％～78.65±9.33％)MC灰度值显著(p＜0.05)降低。脱颗粒是MC释放介质过程，而灰度值的变化可反映颗粒浓度的变化，这表明肉鸡PHS形成过程中MC释放介质增多。(3)本研究还发现，低温组肉鸡组织中的MC数量减少，其中肺血管壁(6.40±1.29～15.67±2.56 cells／mm~2 vs 27.56±1.16～38.51±2.90 cells／mm~2)、肺血管腔(10.59±0.58～19.06±0.90 cells／mm~2 vs 37.55±6.39～62.95±11.74 cells／mm~2)、气管粘膜(105.21±28.74～186.48±18.86 cells／mm~2 vs 230.18±6.49～291.73±30.25cells／mm~2)和心脏(7.47±1.95～10.39±1.99 cells／mm~2 vs 22.08±2.57～41.17±3.45 cells／mm~2)中的MC数均显著(p＜0.05)或极显著(p＜0.01)低于常温组。这些结果表明，环境低温诱发PHS肉鸡肺动脉高压、肺血管重塑、右心收缩和舒张功能及右心肥大等可能与环境低温诱发PHS肉鸡肺血管壁、血管腔、气管粘膜和心肌中MC数量的减少、脱颗粒率的增高和平均灰度值的降低等生物学特性的改变有关。
2．利用焦锑酸钾组化电镜技术，观察肺组织内MC细胞脱颗粒情况和MC脱颗粒时胞浆中Ca~(2+)含量的变化，以确定MC的激活是否有钙超载现象。慢性给予钙通道阻断剂(维拉帕米)后对肉鸡血流动力学、右心功能和MC的影响。结果显示，(1)PHS肉鸡的脱颗粒的MC胞浆中可见Ca~(2+)颗粒沉积，出现Ca~(2+)超载现象。(2)维拉帕米组肺血管壁MC数(13.26±1.57～21.91±1.71 cells／mm~2)显著(p＜0.05)高于低温组(6.40±1.29～15.67+2.56 cells／mm~2)，心脏MC数(36.10±2.90～43.12±2.58cells／mm~2)极显著(p＜0.01)高于低温组(7.47±1.95～8.80±1.48 cells／mm~2)；维拉帕米组肺实质和肺小动脉血管壁MC脱颗粒率也显著(p＜0.05)低于低温组。同时，维拉帕米组肉鸡的肺动脉收
缩压(2.52土o斜、.3.9x士。悦冲a)显著(严0‘05)低于低温组(3.89土0.3如5.17士0.42好a)、舒张
压(082士0.42、1.43士043廿a)也显著低于(P<0.05)低温组(1.76土0.44~2.80士0，57沙a);维持
右心收缩功能和舒张功能相对正常，抑制右心肥大。这表明钙信号参与PHS肉鸡MC的激活，
钙通道阻断剂稳定肺血管和心脏中的MC，防止MC脱颗粒、释放介质，同时降低了肺动脉压，
证明MC脱颗粒在肉鸡PHS发生发展具有重要作用。
3.组胺是MC颗粒中主要的胺类介质，为了观察内源性组胺的作用，采用组胺H:和H:受体拮
抗剂(扑尔敏和西咪替丁)进行急性和慢性试验。(1)在急性试验中观察扑尔敏和西咪替丁对正
常肺动脉压的影响，发现扑尔敏以降低肺动脉压为主，而西咪替丁则以升高肺动脉压为主。对己
发生肺动脉高压的肉鸡，两种组胺受体拮抗剂均降低肺动脉压(杯0.05)，提示内源性组胺可促进
肺动脉压升高。(2)在慢性试验中观察对肉鸡血流动力学、右心功能的影响。结果发现，肌注扑尔
敏早期可降低肉鸡肺动脉收缩压〔扑尔敏组:2.53士0.49 kPavs低温组:4.27士0.58 kpa)，肌注
西咪替丁后期降低肺动脉收缩压(西咪替丁:2.37士0.23vs低温组:5.17士0.42沙a)和舒张压(0.61
士016vs2.80士0.57kPa);(3)扑尔敏维持相对正常的右心收缩和舒张功能(}月pldtmax}:53.20
士14.1小79.80二6.28kP眺ec)。西咪替丁使右心收缩和舒张功能处于较低水平(}过n/dtmax}:
33.20士2.10~61，18士3肠呼扔ec)。两种受体拮抗剂均降低了右心肥大(扑尔敏:小14.29%vs小1000/05
西眯替丁:0 vso、71.43%)和右心衰竭发生率(扑尔敏:14.29%vs 85.71%，西米替丁:0)。这表
明两种组胺受体拮抗剂抑制右心肥大和衰蝎，提示内源性组胺促进右心肥大和衰蝎。(
Pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) and failure (RVF) play an important role in the development of pulmonary hypertension syndrome (PHS). Many studies focus on endothelial cells which induced the pulmonary vasoconstrition and remodeling of pulmonary blood vessels in hypoxia pulmonary hypertension , but some previous studies reported that mast cells (MC) also play an important role in those process. The purpose of this experiment was to study the relationship between mast cells and PHS induced by low ambient temperatures in broiler chickens.
1 .The technique for catheterization of right ventricle was used for measuring the pulmonary artery pressure, right ventricular pressure and the maximum increasing rate of right ventricular (RV±dp/dtmax) was similar to that described by Qiao et al. MC was stained with Alcian blue staining method. Image analysis were measured for the area and the average grey of value of MC, and vessel wall area, vessel total area and mean medial thickness in pulmonary arterioles. The rate of degraunlation of MC was calculated. The result shows: (1) At 38 and 45 days of age, some birds of low ambient temperature group (LT) occured ascites (14.28%), it indicates the PHS was induced successfully. At 24, 31,38 days of age, the mean pulmonary artery pressure (mPAP) of LT (2.47 ± 0.40-3.59 ± 0.47 kPa) was significantly higher than that of normal temperature group(NTX 1.07±0.02-2.76±0.59 kPa). The right ventricular systolic and diastolic function decreased significantly ( p <0.05). The ascites heart index (AHI) of LT was significan
tly more than that of NT at 45 days of age (p<0.05). (2) The rate of degranuation of MC of pulmonary parenchyma (17.49±1.55%~35.14±3.66% vs 9.91±0.87%-18.61 ± 1.76%), peripheral pulmonary blood vessel walls (PPBVWX12.32±1.65%- 21.77±3.68 % vs 35.28 ±2.90%-44.77±2.78%) of lung in LT were more significantly than in NT(/t<0.05). The average grey of value of MC were decreased significantly in LT ( pulmonary parenchyma: 115.44±16.24%~128.48 ± 11.19 % vs 72.89±12.25%~83.33 ±11.85%, PPBVW: 115.68±12.97%~129.97±14.01% vs 67.15 ±10.62%~78.65±9.33%) than in NT. (3) the MC number of peripheral pulmonary blood vessel walls (6.40±1.29-15.67±2.56 cells/mm2 vs 27.56± 1.16-38.51±2.90 cells/mm2), pulmonary inner blood vessel (10.59 ± 0.58-19.06 ± 0.90 cells/mm2 vs 37.55 ± 6.39-62.95 ± 11.74 cells/mm2) , mucosal bronchus (105.21±28.74-186.48±18.86 cells/mm2 vs 230.18±6.49-291.73±30.25 cells/mm2) and right ventricle (7.47±1.95-10.39 ±1.99 cells/mm2 vs 22.08±2.57- 41.17 ± 3.45 cells/mm2) were decreased significantly ( p <0.05).
The change of biological properties of MC may be caused by released the granule mediator during the development of PHS, it shows that the MC could be involved in the development of PHS.
2. The transmembrane movement of Ca 2± was observed in MC degraunlated by LSCM. The result shows: (1) Degraunlated MC was increased significantly intracellular Ca2±. (2)The Ca2± channel
blocking agent (verapamil) can decrease significantly the pulmonary artery systolic pressure (PASP) (2.52±0.84-3.91±0.62 kPa vs 3.89±0.34~5.17±0.42 kPa) and pulmonary artery diastolic pressure (PADP) (0.82±0.42-1.43±0.43 kPa vs 1.76±0.44-2.80 ±0.57 kPa), and promote the number of MC in peripheral pulmonary blood vessel walls (13.26±1.57~21.91±1.71 cells/mm2 vs 6.40±1.29~15.67±2.56 cells/mm2) and right ventricle (36.10±2.90~43.12±2.58 cells/mm2 vs 7.47±1.95~8.80±1.48 cells/mm2), and decrease markedly the rate of degranulation of MC. This result suggests that the degranulation of MC could be related to the development of PHS.
3. This paper investigated the effect of two histamine receptor antagonists, chlorphenamine maleate (Hi-receptors antagonists) and cixnetidine (H2-receptors antagonists), on PHS induced by low ambient temperature in acute and chronic experiment in broiler chickens. The results were as follows: (1) In acute trial, chlorphenamine maleate decreased the PASP (2.7
1．以环境低温诱发肉鸡PHS，测定各组肺动脉压、股动脉压、右心室压、右心室内压最大变化速率等指标的变化，并对肺血管重塑进行观察，同时对肺实质、肺血管壁、肺血管腔、支气管粘膜、心脏和肝脏中的MC数、MC面积、灰度值进行研究。结果显示，(1)环境低温诱发了肉鸡肺动脉高压综合征，低温组形成肺动脉高压(低温组平均肺动脉压2.47±0.40～3.59±0.47kPa vs常温组1.07±0.02～2.76±0.59kPa)，右心肥大(AHI高达32.53±4.78％)，腹水发生率为14.28％。(2)低温组在肺动脉高压形成、右心肥大及衰竭过程中，肺实质(17.49±1.55％～35.14±3.66％vs9.91±0.87％～18.61±1.76％)、肺血管壁MC脱颗粒率(12.32±1.65％～21.77±3.68％vs 35.28±2.90％～44.77±2.78％)显著(p＜0.05)升高，肺实质(115.44±16.24％～128.48±11.19％vs 72.89±12.25％～83.33±11.85％)、肺血管壁(115.68±12.97％～129.97±14.01％vs 67.15±10.62％～78.65±9.33％)MC灰度值显著(p＜0.05)降低。脱颗粒是MC释放介质过程，而灰度值的变化可反映颗粒浓度的变化，这表明肉鸡PHS形成过程中MC释放介质增多。(3)本研究还发现，低温组肉鸡组织中的MC数量减少，其中肺血管壁(6.40±1.29～15.67±2.56 cells／mm~2 vs 27.56±1.16～38.51±2.90 cells／mm~2)、肺血管腔(10.59±0.58～19.06±0.90 cells／mm~2 vs 37.55±6.39～62.95±11.74 cells／mm~2)、气管粘膜(105.21±28.74～186.48±18.86 cells／mm~2 vs 230.18±6.49～291.73±30.25cells／mm~2)和心脏(7.47±1.95～10.39±1.99 cells／mm~2 vs 22.08±2.57～41.17±3.45 cells／mm~2)中的MC数均显著(p＜0.05)或极显著(p＜0.01)低于常温组。这些结果表明，环境低温诱发PHS肉鸡肺动脉高压、肺血管重塑、右心收缩和舒张功能及右心肥大等可能与环境低温诱发PHS肉鸡肺血管壁、血管腔、气管粘膜和心肌中MC数量的减少、脱颗粒率的增高和平均灰度值的降低等生物学特性的改变有关。
2．利用焦锑酸钾组化电镜技术，观察肺组织内MC细胞脱颗粒情况和MC脱颗粒时胞浆中Ca~(2+)含量的变化，以确定MC的激活是否有钙超载现象。慢性给予钙通道阻断剂(维拉帕米)后对肉鸡血流动力学、右心功能和MC的影响。结果显示，(1)PHS肉鸡的脱颗粒的MC胞浆中可见Ca~(2+)颗粒沉积，出现Ca~(2+)超载现象。(2)维拉帕米组肺血管壁MC数(13.26±1.57～21.91±1.71 cells／mm~2)显著(p＜0.05)高于低温组(6.40±1.29～15.67+2.56 cells／mm~2)，心脏MC数(36.10±2.90～43.12±2.58cells／mm~2)极显著(p＜0.01)高于低温组(7.47±1.95～8.80±1.48 cells／mm~2)；维拉帕米组肺实质和肺小动脉血管壁MC脱颗粒率也显著(p＜0.05)低于低温组。同时，维拉帕米组肉鸡的肺动脉收
缩压(2.52土o斜、.3.9x士。悦冲a)显著(严0‘05)低于低温组(3.89土0.3如5.17士0.42好a)、舒张
压(082士0.42、1.43士043廿a)也显著低于(P<0.05)低温组(1.76土0.44~2.80士0，57沙a);维持
右心收缩功能和舒张功能相对正常，抑制右心肥大。这表明钙信号参与PHS肉鸡MC的激活，
钙通道阻断剂稳定肺血管和心脏中的MC，防止MC脱颗粒、释放介质，同时降低了肺动脉压，
证明MC脱颗粒在肉鸡PHS发生发展具有重要作用。
3.组胺是MC颗粒中主要的胺类介质，为了观察内源性组胺的作用，采用组胺H:和H:受体拮
抗剂(扑尔敏和西咪替丁)进行急性和慢性试验。(1)在急性试验中观察扑尔敏和西咪替丁对正
常肺动脉压的影响，发现扑尔敏以降低肺动脉压为主，而西咪替丁则以升高肺动脉压为主。对己
发生肺动脉高压的肉鸡，两种组胺受体拮抗剂均降低肺动脉压(杯0.05)，提示内源性组胺可促进
肺动脉压升高。(2)在慢性试验中观察对肉鸡血流动力学、右心功能的影响。结果发现，肌注扑尔
敏早期可降低肉鸡肺动脉收缩压〔扑尔敏组:2.53士0.49 kPavs低温组:4.27士0.58 kpa)，肌注
西咪替丁后期降低肺动脉收缩压(西咪替丁:2.37士0.23vs低温组:5.17士0.42沙a)和舒张压(0.61
士016vs2.80士0.57kPa);(3)扑尔敏维持相对正常的右心收缩和舒张功能(}月pldtmax}:53.20
士14.1小79.80二6.28kP眺ec)。西咪替丁使右心收缩和舒张功能处于较低水平(}过n/dtmax}:
33.20士2.10~61，18士3肠呼扔ec)。两种受体拮抗剂均降低了右心肥大(扑尔敏:小14.29%vs小1000/05
西眯替丁:0 vso、71.43%)和右心衰竭发生率(扑尔敏:14.29%vs 85.71%，西米替丁:0)。这表
明两种组胺受体拮抗剂抑制右心肥大和衰蝎，提示内源性组胺促进右心肥大和衰蝎。(
Pulmonary hypertension (PH) and right ventricular hypertrophy (RVH) and failure (RVF) play an important role in the development of pulmonary hypertension syndrome (PHS). Many studies focus on endothelial cells which induced the pulmonary vasoconstrition and remodeling of pulmonary blood vessels in hypoxia pulmonary hypertension , but some previous studies reported that mast cells (MC) also play an important role in those process. The purpose of this experiment was to study the relationship between mast cells and PHS induced by low ambient temperatures in broiler chickens.
1 .The technique for catheterization of right ventricle was used for measuring the pulmonary artery pressure, right ventricular pressure and the maximum increasing rate of right ventricular (RV±dp/dtmax) was similar to that described by Qiao et al. MC was stained with Alcian blue staining method. Image analysis were measured for the area and the average grey of value of MC, and vessel wall area, vessel total area and mean medial thickness in pulmonary arterioles. The rate of degraunlation of MC was calculated. The result shows: (1) At 38 and 45 days of age, some birds of low ambient temperature group (LT) occured ascites (14.28%), it indicates the PHS was induced successfully. At 24, 31,38 days of age, the mean pulmonary artery pressure (mPAP) of LT (2.47 ± 0.40-3.59 ± 0.47 kPa) was significantly higher than that of normal temperature group(NTX 1.07±0.02-2.76±0.59 kPa). The right ventricular systolic and diastolic function decreased significantly ( p <0.05). The ascites heart index (AHI) of LT was significan
tly more than that of NT at 45 days of age (p<0.05). (2) The rate of degranuation of MC of pulmonary parenchyma (17.49±1.55%~35.14±3.66% vs 9.91±0.87%-18.61 ± 1.76%), peripheral pulmonary blood vessel walls (PPBVWX12.32±1.65%- 21.77±3.68 % vs 35.28 ±2.90%-44.77±2.78%) of lung in LT were more significantly than in NT(/t<0.05). The average grey of value of MC were decreased significantly in LT ( pulmonary parenchyma: 115.44±16.24%~128.48 ± 11.19 % vs 72.89±12.25%~83.33 ±11.85%, PPBVW: 115.68±12.97%~129.97±14.01% vs 67.15 ±10.62%~78.65±9.33%) than in NT. (3) the MC number of peripheral pulmonary blood vessel walls (6.40±1.29-15.67±2.56 cells/mm2 vs 27.56± 1.16-38.51±2.90 cells/mm2), pulmonary inner blood vessel (10.59 ± 0.58-19.06 ± 0.90 cells/mm2 vs 37.55 ± 6.39-62.95 ± 11.74 cells/mm2) , mucosal bronchus (105.21±28.74-186.48±18.86 cells/mm2 vs 230.18±6.49-291.73±30.25 cells/mm2) and right ventricle (7.47±1.95-10.39 ±1.99 cells/mm2 vs 22.08±2.57- 41.17 ± 3.45 cells/mm2) were decreased significantly ( p <0.05).
The change of biological properties of MC may be caused by released the granule mediator during the development of PHS, it shows that the MC could be involved in the development of PHS.
2. The transmembrane movement of Ca 2± was observed in MC degraunlated by LSCM. The result shows: (1) Degraunlated MC was increased significantly intracellular Ca2±. (2)The Ca2± channel
blocking agent (verapamil) can decrease significantly the pulmonary artery systolic pressure (PASP) (2.52±0.84-3.91±0.62 kPa vs 3.89±0.34~5.17±0.42 kPa) and pulmonary artery diastolic pressure (PADP) (0.82±0.42-1.43±0.43 kPa vs 1.76±0.44-2.80 ±0.57 kPa), and promote the number of MC in peripheral pulmonary blood vessel walls (13.26±1.57~21.91±1.71 cells/mm2 vs 6.40±1.29~15.67±2.56 cells/mm2) and right ventricle (36.10±2.90~43.12±2.58 cells/mm2 vs 7.47±1.95~8.80±1.48 cells/mm2), and decrease markedly the rate of degranulation of MC. This result suggests that the degranulation of MC could be related to the development of PHS.
3. This paper investigated the effect of two histamine receptor antagonists, chlorphenamine maleate (Hi-receptors antagonists) and cixnetidine (H2-receptors antagonists), on PHS induced by low ambient temperature in acute and chronic experiment in broiler chickens. The results were as follows: (1) In acute trial, chlorphenamine maleate decreased the PASP (2.7
引文
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