钙敏感受体在高肺血流性肺动脉高压大鼠的表达及其在内质网应激中的作用
发布时间：2018-09-17 14:38
【摘要】：第一章高肺血流性肺动脉高压大鼠模型的建立 研究目的 观察比较左肺切除术与腹主动脉-下腔静脉分流术两种方法所造的大鼠肺动脉高压模型在血流动力学和肺血管形态学方面的病理生理变化和差异,选择合适的动物模型进行进一步的研究。 材料与方法 160只SD大鼠随机为正常对照组(Control,n=40)、左肺切除组(PE,n=60)和腹主动脉-下腔静脉分流组(A-V,n=60),于第1周、第2周、第4周、第6周分别观察各项形态学指标。正常对照组每个亚组10只,PE组和A-V组每个亚组15只。各组大鼠于广西医科大学动物实验中心(SPF级)饲养至指定时间进行观察。分别测定各组大鼠肺动脉平均压(mPAP),检测右心室/左心室+室间隔(right ventricle/left ventricle plus septum,RV/LV+S)比值；常规石蜡包埋大鼠肺组织,切片后做HE染色,观察各组大鼠肺组织中小动脉的形态变化,计算管壁厚度占外径的百分比(WT%)及管壁面积占血管总面积的百分比(WA%),作为反映血管重构程度的指标。 结果 (1)模型建造过程中,A-V组大鼠死亡例数比PE组大鼠死亡数明显要低,手术技巧及术后感染是PE组大鼠死亡的最主要原因。 (2)PE组和A-V组mPAP术后出现较为相似的走高趋势：术后1W有明显的增高,至第2W出现下探,4W后开始逐渐回升。与对照组比较,A-V组除2W组降至正常值附近外,1W、4W和6W组大鼠1mPAP均显著性升高(P0.05)；尽管2周组也出现mPAP下探,但PE组术后各个时间点(1W、2W、4W和6W)mPAP均较对照组明显升高(P0.05)。PE组术后mPAP各个时间点均较A-V组各时间点要明显增高(P0.05)。 (3)随着术后时间的延长,A-V组和PE组的右心室肥厚指数(RVHI)均进行性增高：与对照组比较,A-V组和PE组术后各个时间点的RVHI显著性升高(P0.05)。与A-V组比较,PE组RVHI明显升高(P0.05)。 (4)随着术后时间的延长,A-V组和PE组WT%均呈现增高趋势：从术后2W开始,A-V组和PE组WT%均较正常对照组显著上升(P0.05)；从术后2W开始,PE组WT%较A-V组明显增高(P0.05)。 (5)随着术后时间的延长,A-V组和PE组WA%均呈现进行性增高趋势：与对照组比较,A-V组和PE组WA%从术后2W开始显著增高(P0.05)；术后4W开始,PE组WA%较A-V组显著增高(P0.05)。 结论 (1)大鼠左肺切除和腹主动脉-下腔静脉分流肺动脉高压模型术后均出现了右心肥厚和肺动脉高压的临床表现,表明两种模型均有效地模拟了早期先天性心脏病相关性肺动脉高压的血管形态学及病理生理的改变,发生了以中膜增生肥厚和管腔狭窄,管壁厚度增加为特征的组织病理学改变。两种模型均可用于研究先天性心脏病相关性肺动脉高压的可靠、有效的动物模型。 (2)与腹主动脉-下腔静脉分流肺动脉高压模型比较,大鼠左肺切除肺动脉高压模型造模周期更短,血流动力学改变更明显,肺血管形态学改变更严重等优点。 第二章钙敏感受体在高肺血流性肺动脉高压大鼠的表达 研究目的 观察钙敏感受体(CaSR)在高肺血流性肺动脉高压大鼠肺动脉平滑肌的表达,并探讨其在高肺血流性肺动脉高压形成的表达情况。 材料与方法 行左肺切除手术建立大鼠肺动脉高压模型,将27只大鼠随机分为3组(n=9),对照组,手术组,手术+钙敏感受体阻滞剂Calhex231组。术后饲养35d,分别测定各组大鼠肺动脉平均压(mPAP),计算右心室/体重(right ventricle/body weight, RV/BW)和右心室肥厚指数(RVHI)。应用实时定量PCR检测各组大鼠肺动脉CaSRmRNA的表达,HE染色观察肺动脉结构的改变。 结果 (1)与对照组比较,左肺切除术后35d时,手术组大鼠mPAP、RV/BW和RVHI均明显高于对照组,差异有统计学意义,说明用左肺切除术造肺动脉高压模型成功；与手术组比,手术+Calhex231组大鼠mPAP、RV/BW和RVHI均明显降低(P0.05)。 (2)与对照组比较,手术组大鼠肺动脉CaSRmRNA的表达明显升高(P0.05)；与手术组比较,手术+Calhex231组大鼠肺动脉CaSRmRNA的表达明显降低(P0.05)。 结论 高肺血流量上调了CaSR的表达；CaSR通过诱导肺血管增殖与重构参与了高肺血流性肺动脉高压发生的过程。 第三章钙敏感受体在高肺血流性肺动脉高压大鼠内质网应激的作用研究目的 内质网应激(endoplasmic reticulum stress,ERS)作为众多心血管疾病发生发展的重要病理机制,近年来日益受到人们的广泛关注。既往研究认为,CaSR能够通过激活内质网应激增加组织的损伤程度,但CaSR与ERS在高肺血流性肺动脉高压的发生发展过程中有无关系及其具体机制尚不清楚。本实验以高肺血流性肺动脉高压大鼠为研究对象,探讨CaSR对高肺血流性肺动脉高压大鼠内质网应激的影响,进而对其机制做进一步研究。 材料与方法 行左肺切除手术建立大鼠肺动脉高压模型,将27只大鼠随机分为3组(n=9),对照组,手术组,手术+钙敏感受体阻滞剂Calhex231组。术后饲养35天,采用原位缺口末端标记(TUNEL)方法检测内皮细胞的凋亡,采用免疫组织化学染色法检测肺动脉组织中内质网应激标志糖调节蛋白78(GRP78)、内质网相关性凋亡标志蛋白半胱天冬水解酶-12(caspasel2)和磷酸化的真核细胞蛋白质翻译起始复合体(p-eIF2a)的蛋白表达,并用全自动图像分析系统对其进行半定量分析。 结果 (1)左肺切除术后35d时,各组大鼠肺动脉内皮细胞中均存在凋亡细胞。与对照组比较,手术组肺动脉内皮细胞凋亡率明显增高(P0.05)；与手术组比较,手术+Calhex231组肺动脉内皮细胞凋亡率明显降低(P0.05)。 (2)与对照组相比,手术组大鼠肺动脉GRP78、caspase12和p-eIF2a表达均明显增高(P0.05)；与手术组相比,手术+Calhex231组大鼠肺动脉GRP78、 caspase12和p-eIF2a表达明显降低(P0.05)。 结论 左肺切除术后35天所致高肺血流性肺动脉高压大鼠肺动脉中内质网应激显著增强。CaSR可通过激活过度内质网应激反应促进高肺血流性肺动脉高压和肺血管结构重建形成。
[Abstract]:Chapter 1 Establishment of a rat model of pulmonary hypertension with high pulmonary blood flow
research objective
To observe and compare the pathophysiological changes and differences in hemodynamics and pulmonary vascular morphology between left pulmonary resection and abdominal aortic-inferior vena cava shunt in rats with pulmonary hypertension.
Materials and methods
160 SD rats were randomly divided into normal control group (Control, n = 40), left pulmonary resection group (PE, n = 60) and abdominal aortic-inferior vena cava shunt group (A-V, n = 60). Morphological indexes were observed in the first week, the second week, the fourth week and the sixth week. The pulmonary artery mean pressure (mPAP), right ventricle / left ventricle plus septum (RV / LV + S) ratio and the shape of small arteries in lung tissue of rats were observed by HE staining after paraffin embedding. The percentage of wall thickness to external diameter (WT%) and the percentage of wall area to total vascular area (WA%) were calculated as indicators of vascular remodeling.
Result
(1) The death rate of rats in A-V group was significantly lower than that in PE group during the process of model construction. Surgical skills and postoperative infection were the main causes of death in PE group.
(2) The mPAP of PE group and A-V group showed a similar trend after operation: the mPAP of PE group increased significantly at 1W, descended at 2W, and began to rise gradually after 4W. Compared with the control group, except for the descent of 2W group, the mPAP of 1W, 4W and 6W groups increased significantly (P 0.05); although the descent of mPAP was also found in 2-week group, the mPAP of PE group increased significantly after operation. Time point (1W, 2W, 4W and 6W) mPAP was significantly higher than that of the control group (P 0.05). After operation, the mPAP in PE group was significantly higher than that in A-V group (P 0.05).
(3) With the prolongation of postoperative time, the right ventricular hypertrophy index (RVHI) of A-V group and PE group increased progressively: Compared with the control group, the RVHI of A-V group and PE group increased significantly at each time point after operation (P 0.05). Compared with A-V group, the RVHI of PE group increased significantly (P 0.05).
(4) With the prolongation of postoperative time, WT% of A-V group and PE group showed an increasing trend: WT% of A-V group and PE group were significantly higher than that of normal control group from 2W after operation (P 0.05); WT% of PE group was significantly higher than that of A-V group from 2W after operation (P 0.05).
(5) With the prolongation of postoperative time, WA% of A-V group and PE group showed a progressive increase trend: compared with the control group, WA% of A-V group and PE group increased significantly from 2W after surgery (P 0.05); from 4W after surgery, WA% of PE group was significantly higher than A-V group (P 0.05).
conclusion
(1) Right heart hypertrophy and pulmonary hypertension were found in both left pulmonary resection and abdominal aortic-inferior vena cava shunt pulmonary hypertension models in rats. The results showed that both models could effectively simulate the changes of vascular morphology and pathophysiology of pulmonary hypertension associated with early congenital heart disease, and the hypertrophy of pulmonary artery was induced by mesangial hyperplasia. Thickness, lumen stenosis, and increased wall thickness are characteristic histopathological changes. Both models can be used to study reliable and effective animal models of congenital heart disease-associated pulmonary hypertension.
(2) Compared with the pulmonary hypertension model of abdominal aorta-inferior vena cava shunt, the left pulmonary resection pulmonary hypertension model had shorter modeling cycle, more obvious hemodynamic changes and more serious morphological changes of pulmonary vessels.
The second chapter is the expression of calcium sensing receptor in rats with pulmonary hypertension induced by pulmonary hypertension.
research objective
To observe the expression of calcium-sensitive receptor (CaSR) in pulmonary artery smooth muscle of rats with high pulmonary blood flow pulmonary hypertension and to explore its expression in the formation of high pulmonary blood flow pulmonary hypertension.
Materials and methods
Twenty-seven rats were randomly divided into three groups (n=9), control group, operation group, operation plus calcium-sensitive receptor blocker Calhex 231. After 35 days of feeding, the mean pulmonary artery pressure (mPAP) and right ventricular/body weight (RV/BW) were measured and the right ventricular hypertrophy index (RV/BW) were calculated. Real-time quantitative PCR was used to detect the expression of CaSR mRNA in pulmonary artery of rats in each group, and HE staining was used to observe the changes of pulmonary artery structure.
Result
(1) Compared with the control group, the mPAP, RV/BW and RVHI in the operation group were significantly higher than those in the control group at 35 days after left pneumonectomy, and the difference was statistically significant, indicating that the pulmonary hypertension model was successfully established by left pneumonectomy; compared with the operation group, the mPAP, RV/BW and RVHI in the operation + Calhex231 group were significantly decreased (P 0.05).
(2) Compared with the control group, the expression of CaSR mRNA in the pulmonary artery of the rats in the operation group increased significantly (P 0.05), and the expression of CaSR mRNA in the pulmonary artery of the rats in the operation + Calhex 231 group decreased significantly (P 0.05).
conclusion
High pulmonary blood flow increased the expression of CaSR, and CaSR participated in the development of high pulmonary blood flow pulmonary hypertension by inducing pulmonary vascular proliferation and remodeling.
The third chapter is about the role of calcium sensing receptor in endoplasmic reticulum stress in rats with pulmonary hypertension.
Endoplasmic reticulum stress (ERS), as an important pathological mechanism in the development of many cardiovascular diseases, has attracted more and more attention in recent years. In this study, we investigated the effects of CaSR on endoplasmic reticulum (ER) stress in rats with high pulmonary blood flow pulmonary hypertension (HPPH).
Materials and methods
Twenty-seven rats were randomly divided into 3 groups (n=9), control group, operation group, operation + calcium-sensitive receptor blocker Calhex 231 group. After 35 days of feeding, the apoptosis of endothelial cells was detected by in situ nick end labeling (TUNEL), and the pulmonary artery tissues were detected by immunohistochemical staining. The expression of glucose-regulated protein 78 (GRP78), caspase-2 (caspase-2), and phosphorylated eukaryotic protein translation initiation complex (p-eIF2a), a stress marker of endoplasmic reticulum (ER), was analyzed by automatic image analysis system.
Result
(1) There were apoptotic cells in pulmonary artery endothelial cells 35 days after left pulmonary resection. Compared with the control group, the apoptotic rate of pulmonary artery endothelial cells in the operation group was significantly higher (P 0.05), and the apoptotic rate of pulmonary artery endothelial cells in the operation + Calhex 231 group was significantly lower (P 0.05).
(2) Compared with the control group, the expressions of GRP78, caspase 12 and p-eIF2a in the pulmonary artery of the rats in the operation group were significantly higher (P 0.05), and the expressions of GRP78, caspase 12 and p-eIF2a in the pulmonary artery of the rats in the operation + Calhex 231 group were significantly lower (P 0.05).
conclusion
Endoplasmic reticulum stress in the pulmonary artery of rats with high pulmonary blood flow pulmonary hypertension was significantly increased 35 days after left pulmonary resection. CaSR could promote the formation of high pulmonary blood flow pulmonary hypertension and pulmonary vascular remodeling by activating excessive endoplasmic reticulum stress response.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R655.3
[Abstract]:Chapter 1 Establishment of a rat model of pulmonary hypertension with high pulmonary blood flow
research objective
To observe and compare the pathophysiological changes and differences in hemodynamics and pulmonary vascular morphology between left pulmonary resection and abdominal aortic-inferior vena cava shunt in rats with pulmonary hypertension.
Materials and methods
160 SD rats were randomly divided into normal control group (Control, n = 40), left pulmonary resection group (PE, n = 60) and abdominal aortic-inferior vena cava shunt group (A-V, n = 60). Morphological indexes were observed in the first week, the second week, the fourth week and the sixth week. The pulmonary artery mean pressure (mPAP), right ventricle / left ventricle plus septum (RV / LV + S) ratio and the shape of small arteries in lung tissue of rats were observed by HE staining after paraffin embedding. The percentage of wall thickness to external diameter (WT%) and the percentage of wall area to total vascular area (WA%) were calculated as indicators of vascular remodeling.
Result
(1) The death rate of rats in A-V group was significantly lower than that in PE group during the process of model construction. Surgical skills and postoperative infection were the main causes of death in PE group.
(2) The mPAP of PE group and A-V group showed a similar trend after operation: the mPAP of PE group increased significantly at 1W, descended at 2W, and began to rise gradually after 4W. Compared with the control group, except for the descent of 2W group, the mPAP of 1W, 4W and 6W groups increased significantly (P 0.05); although the descent of mPAP was also found in 2-week group, the mPAP of PE group increased significantly after operation. Time point (1W, 2W, 4W and 6W) mPAP was significantly higher than that of the control group (P 0.05). After operation, the mPAP in PE group was significantly higher than that in A-V group (P 0.05).
(3) With the prolongation of postoperative time, the right ventricular hypertrophy index (RVHI) of A-V group and PE group increased progressively: Compared with the control group, the RVHI of A-V group and PE group increased significantly at each time point after operation (P 0.05). Compared with A-V group, the RVHI of PE group increased significantly (P 0.05).
(4) With the prolongation of postoperative time, WT% of A-V group and PE group showed an increasing trend: WT% of A-V group and PE group were significantly higher than that of normal control group from 2W after operation (P 0.05); WT% of PE group was significantly higher than that of A-V group from 2W after operation (P 0.05).
(5) With the prolongation of postoperative time, WA% of A-V group and PE group showed a progressive increase trend: compared with the control group, WA% of A-V group and PE group increased significantly from 2W after surgery (P 0.05); from 4W after surgery, WA% of PE group was significantly higher than A-V group (P 0.05).
conclusion
(1) Right heart hypertrophy and pulmonary hypertension were found in both left pulmonary resection and abdominal aortic-inferior vena cava shunt pulmonary hypertension models in rats. The results showed that both models could effectively simulate the changes of vascular morphology and pathophysiology of pulmonary hypertension associated with early congenital heart disease, and the hypertrophy of pulmonary artery was induced by mesangial hyperplasia. Thickness, lumen stenosis, and increased wall thickness are characteristic histopathological changes. Both models can be used to study reliable and effective animal models of congenital heart disease-associated pulmonary hypertension.
(2) Compared with the pulmonary hypertension model of abdominal aorta-inferior vena cava shunt, the left pulmonary resection pulmonary hypertension model had shorter modeling cycle, more obvious hemodynamic changes and more serious morphological changes of pulmonary vessels.
The second chapter is the expression of calcium sensing receptor in rats with pulmonary hypertension induced by pulmonary hypertension.
research objective
To observe the expression of calcium-sensitive receptor (CaSR) in pulmonary artery smooth muscle of rats with high pulmonary blood flow pulmonary hypertension and to explore its expression in the formation of high pulmonary blood flow pulmonary hypertension.
Materials and methods
Twenty-seven rats were randomly divided into three groups (n=9), control group, operation group, operation plus calcium-sensitive receptor blocker Calhex 231. After 35 days of feeding, the mean pulmonary artery pressure (mPAP) and right ventricular/body weight (RV/BW) were measured and the right ventricular hypertrophy index (RV/BW) were calculated. Real-time quantitative PCR was used to detect the expression of CaSR mRNA in pulmonary artery of rats in each group, and HE staining was used to observe the changes of pulmonary artery structure.
Result
(1) Compared with the control group, the mPAP, RV/BW and RVHI in the operation group were significantly higher than those in the control group at 35 days after left pneumonectomy, and the difference was statistically significant, indicating that the pulmonary hypertension model was successfully established by left pneumonectomy; compared with the operation group, the mPAP, RV/BW and RVHI in the operation + Calhex231 group were significantly decreased (P 0.05).
(2) Compared with the control group, the expression of CaSR mRNA in the pulmonary artery of the rats in the operation group increased significantly (P 0.05), and the expression of CaSR mRNA in the pulmonary artery of the rats in the operation + Calhex 231 group decreased significantly (P 0.05).
conclusion
High pulmonary blood flow increased the expression of CaSR, and CaSR participated in the development of high pulmonary blood flow pulmonary hypertension by inducing pulmonary vascular proliferation and remodeling.
The third chapter is about the role of calcium sensing receptor in endoplasmic reticulum stress in rats with pulmonary hypertension.
Endoplasmic reticulum stress (ERS), as an important pathological mechanism in the development of many cardiovascular diseases, has attracted more and more attention in recent years. In this study, we investigated the effects of CaSR on endoplasmic reticulum (ER) stress in rats with high pulmonary blood flow pulmonary hypertension (HPPH).
Materials and methods
Twenty-seven rats were randomly divided into 3 groups (n=9), control group, operation group, operation + calcium-sensitive receptor blocker Calhex 231 group. After 35 days of feeding, the apoptosis of endothelial cells was detected by in situ nick end labeling (TUNEL), and the pulmonary artery tissues were detected by immunohistochemical staining. The expression of glucose-regulated protein 78 (GRP78), caspase-2 (caspase-2), and phosphorylated eukaryotic protein translation initiation complex (p-eIF2a), a stress marker of endoplasmic reticulum (ER), was analyzed by automatic image analysis system.
Result
(1) There were apoptotic cells in pulmonary artery endothelial cells 35 days after left pulmonary resection. Compared with the control group, the apoptotic rate of pulmonary artery endothelial cells in the operation group was significantly higher (P 0.05), and the apoptotic rate of pulmonary artery endothelial cells in the operation + Calhex 231 group was significantly lower (P 0.05).
(2) Compared with the control group, the expressions of GRP78, caspase 12 and p-eIF2a in the pulmonary artery of the rats in the operation group were significantly higher (P 0.05), and the expressions of GRP78, caspase 12 and p-eIF2a in the pulmonary artery of the rats in the operation + Calhex 231 group were significantly lower (P 0.05).
conclusion
Endoplasmic reticulum stress in the pulmonary artery of rats with high pulmonary blood flow pulmonary hypertension was significantly increased 35 days after left pulmonary resection. CaSR could promote the formation of high pulmonary blood flow pulmonary hypertension and pulmonary vascular remodeling by activating excessive endoplasmic reticulum stress response.
【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R655.3
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