【摘要】：第一章高肺血流性肺動(dòng)脈高壓大鼠模型的建立 研究目的 觀察比較左肺切除術(shù)與腹主動(dòng)脈-下腔靜脈分流術(shù)兩種方法所造的大鼠肺動(dòng)脈高壓模型在血流動(dòng)力學(xué)和肺血管形態(tài)學(xué)方面的病理生理變化和差異,選擇合適的動(dòng)物模型進(jìn)行進(jìn)一步的研究。 材料與方法 160只SD大鼠隨機(jī)為正常對(duì)照組(Control,n=40)、左肺切除組(PE,n=60)和腹主動(dòng)脈-下腔靜脈分流組(A-V,n=60),于第1周、第2周、第4周、第6周分別觀察各項(xiàng)形態(tài)學(xué)指標(biāo)。正常對(duì)照組每個(gè)亞組10只,PE組和A-V組每個(gè)亞組15只。各組大鼠于廣西醫(yī)科大學(xué)動(dòng)物實(shí)驗(yàn)中心(SPF級(jí))飼養(yǎng)至指定時(shí)間進(jìn)行觀察。分別測(cè)定各組大鼠肺動(dòng)脈平均壓(mPAP),檢測(cè)右心室/左心室+室間隔(right ventricle/left ventricle plus septum,RV/LV+S)比值；常規(guī)石蠟包埋大鼠肺組織,切片后做HE染色,觀察各組大鼠肺組織中小動(dòng)脈的形態(tài)變化,計(jì)算管壁厚度占外徑的百分比(WT%)及管壁面積占血管總面積的百分比(WA%),作為反映血管重構(gòu)程度的指標(biāo)。 結(jié)果 (1)模型建造過(guò)程中,A-V組大鼠死亡例數(shù)比PE組大鼠死亡數(shù)明顯要低,手術(shù)技巧及術(shù)后感染是PE組大鼠死亡的最主要原因。 (2)PE組和A-V組mPAP術(shù)后出現(xiàn)較為相似的走高趨勢(shì)：術(shù)后1W有明顯的增高,至第2W出現(xiàn)下探,4W后開始逐漸回升。與對(duì)照組比較,A-V組除2W組降至正常值附近外,1W、4W和6W組大鼠1mPAP均顯著性升高(P0.05)；盡管2周組也出現(xiàn)mPAP下探,但PE組術(shù)后各個(gè)時(shí)間點(diǎn)(1W、2W、4W和6W)mPAP均較對(duì)照組明顯升高(P0.05)。PE組術(shù)后mPAP各個(gè)時(shí)間點(diǎn)均較A-V組各時(shí)間點(diǎn)要明顯增高(P0.05)。 (3)隨著術(shù)后時(shí)間的延長(zhǎng),A-V組和PE組的右心室肥厚指數(shù)(RVHI)均進(jìn)行性增高：與對(duì)照組比較,A-V組和PE組術(shù)后各個(gè)時(shí)間點(diǎn)的RVHI顯著性升高(P0.05)。與A-V組比較,PE組RVHI明顯升高(P0.05)。 (4)隨著術(shù)后時(shí)間的延長(zhǎng),A-V組和PE組WT%均呈現(xiàn)增高趨勢(shì)：從術(shù)后2W開始,A-V組和PE組WT%均較正常對(duì)照組顯著上升(P0.05)；從術(shù)后2W開始,PE組WT%較A-V組明顯增高(P0.05)。 (5)隨著術(shù)后時(shí)間的延長(zhǎng),A-V組和PE組WA%均呈現(xiàn)進(jìn)行性增高趨勢(shì)：與對(duì)照組比較,A-V組和PE組WA%從術(shù)后2W開始顯著增高(P0.05)；術(shù)后4W開始,PE組WA%較A-V組顯著增高(P0.05)。 結(jié)論 (1)大鼠左肺切除和腹主動(dòng)脈-下腔靜脈分流肺動(dòng)脈高壓模型術(shù)后均出現(xiàn)了右心肥厚和肺動(dòng)脈高壓的臨床表現(xiàn),表明兩種模型均有效地模擬了早期先天性心臟病相關(guān)性肺動(dòng)脈高壓的血管形態(tài)學(xué)及病理生理的改變,發(fā)生了以中膜增生肥厚和管腔狹窄,管壁厚度增加為特征的組織病理學(xué)改變。兩種模型均可用于研究先天性心臟病相關(guān)性肺動(dòng)脈高壓的可靠、有效的動(dòng)物模型。 (2)與腹主動(dòng)脈-下腔靜脈分流肺動(dòng)脈高壓模型比較,大鼠左肺切除肺動(dòng)脈高壓模型造模周期更短,血流動(dòng)力學(xué)改變更明顯,肺血管形態(tài)學(xué)改變更嚴(yán)重等優(yōu)點(diǎn)。 第二章鈣敏感受體在高肺血流性肺動(dòng)脈高壓大鼠的表達(dá) 研究目的 觀察鈣敏感受體(CaSR)在高肺血流性肺動(dòng)脈高壓大鼠肺動(dòng)脈平滑肌的表達(dá),并探討其在高肺血流性肺動(dòng)脈高壓形成的表達(dá)情況。 材料與方法 行左肺切除手術(shù)建立大鼠肺動(dòng)脈高壓模型,將27只大鼠隨機(jī)分為3組(n=9),對(duì)照組,手術(shù)組,手術(shù)+鈣敏感受體阻滯劑Calhex231組。術(shù)后飼養(yǎng)35d,分別測(cè)定各組大鼠肺動(dòng)脈平均壓(mPAP),計(jì)算右心室/體重(right ventricle/body weight, RV/BW)和右心室肥厚指數(shù)(RVHI)。應(yīng)用實(shí)時(shí)定量PCR檢測(cè)各組大鼠肺動(dòng)脈CaSRmRNA的表達(dá),HE染色觀察肺動(dòng)脈結(jié)構(gòu)的改變。 結(jié)果 (1)與對(duì)照組比較,左肺切除術(shù)后35d時(shí),手術(shù)組大鼠mPAP、RV/BW和RVHI均明顯高于對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義,說(shuō)明用左肺切除術(shù)造肺動(dòng)脈高壓模型成功；與手術(shù)組比,手術(shù)+Calhex231組大鼠mPAP、RV/BW和RVHI均明顯降低(P0.05)。 (2)與對(duì)照組比較,手術(shù)組大鼠肺動(dòng)脈CaSRmRNA的表達(dá)明顯升高(P0.05)；與手術(shù)組比較,手術(shù)+Calhex231組大鼠肺動(dòng)脈CaSRmRNA的表達(dá)明顯降低(P0.05)。 結(jié)論 高肺血流量上調(diào)了CaSR的表達(dá)；CaSR通過(guò)誘導(dǎo)肺血管增殖與重構(gòu)參與了高肺血流性肺動(dòng)脈高壓發(fā)生的過(guò)程。 第三章鈣敏感受體在高肺血流性肺動(dòng)脈高壓大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激的作用研究目的 內(nèi)質(zhì)網(wǎng)應(yīng)激(endoplasmic reticulum stress,ERS)作為眾多心血管疾病發(fā)生發(fā)展的重要病理機(jī)制,近年來(lái)日益受到人們的廣泛關(guān)注。既往研究認(rèn)為,CaSR能夠通過(guò)激活內(nèi)質(zhì)網(wǎng)應(yīng)激增加組織的損傷程度,但CaSR與ERS在高肺血流性肺動(dòng)脈高壓的發(fā)生發(fā)展過(guò)程中有無(wú)關(guān)系及其具體機(jī)制尚不清楚。本實(shí)驗(yàn)以高肺血流性肺動(dòng)脈高壓大鼠為研究對(duì)象,探討CaSR對(duì)高肺血流性肺動(dòng)脈高壓大鼠內(nèi)質(zhì)網(wǎng)應(yīng)激的影響,進(jìn)而對(duì)其機(jī)制做進(jìn)一步研究。 材料與方法 行左肺切除手術(shù)建立大鼠肺動(dòng)脈高壓模型,將27只大鼠隨機(jī)分為3組(n=9),對(duì)照組,手術(shù)組,手術(shù)+鈣敏感受體阻滯劑Calhex231組。術(shù)后飼養(yǎng)35天,采用原位缺口末端標(biāo)記(TUNEL)方法檢測(cè)內(nèi)皮細(xì)胞的凋亡,采用免疫組織化學(xué)染色法檢測(cè)肺動(dòng)脈組織中內(nèi)質(zhì)網(wǎng)應(yīng)激標(biāo)志糖調(diào)節(jié)蛋白78(GRP78)、內(nèi)質(zhì)網(wǎng)相關(guān)性凋亡標(biāo)志蛋白半胱天冬水解酶-12(caspasel2)和磷酸化的真核細(xì)胞蛋白質(zhì)翻譯起始復(fù)合體(p-eIF2a)的蛋白表達(dá),并用全自動(dòng)圖像分析系統(tǒng)對(duì)其進(jìn)行半定量分析。 結(jié)果 (1)左肺切除術(shù)后35d時(shí),各組大鼠肺動(dòng)脈內(nèi)皮細(xì)胞中均存在凋亡細(xì)胞。與對(duì)照組比較,手術(shù)組肺動(dòng)脈內(nèi)皮細(xì)胞凋亡率明顯增高(P0.05)；與手術(shù)組比較,手術(shù)+Calhex231組肺動(dòng)脈內(nèi)皮細(xì)胞凋亡率明顯降低(P0.05)。 (2)與對(duì)照組相比,手術(shù)組大鼠肺動(dòng)脈GRP78、caspase12和p-eIF2a表達(dá)均明顯增高(P0.05)；與手術(shù)組相比,手術(shù)+Calhex231組大鼠肺動(dòng)脈GRP78、 caspase12和p-eIF2a表達(dá)明顯降低(P0.05)。 結(jié)論 左肺切除術(shù)后35天所致高肺血流性肺動(dòng)脈高壓大鼠肺動(dòng)脈中內(nèi)質(zhì)網(wǎng)應(yīng)激顯著增強(qiáng)。CaSR可通過(guò)激活過(guò)度內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)促進(jìn)高肺血流性肺動(dòng)脈高壓和肺血管結(jié)構(gòu)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.
【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R655.3

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