肺血少型先心病幼猪动物模型的建立及肺血改变对未成熟肺血管形态发育和功能的影响

发布时间：2017-12-30 23:32

  本文关键词：肺血少型先心病幼猪动物模型的建立及肺血改变对未成熟肺血管形态发育和功能的影响　出处：《中国协和医科大学》2008年博士论文　论文类型：学位论文

  更多相关文章： 先天性心脏病 紫绀 动物模型 猪 形态定量分析 肺动脉 发育不良 先天性心脏病 紫绀 肺动脉 发育不良 基质金属蛋白酶 组织基质金属蛋白酶抑制剂 血管内皮细胞生长因子 Ⅰ型和Ⅲ型胶原 肺发育

【摘要】： 第一部分.介入和外科技术相结合建立紫绀型肺血少先心病幼猪动物模型 【目的】.采用一种新颖的介入和外科相结合的Hybrid方法建立紫绀型肺血少先心病动物模型。 【方法】.采用生后1～2月的幼猪,体重8～12kg,分为两组,每组8只。a)正常对照组(C-group,n=8):即假手术组,只开胸无手术处理;b)肺血减少组(D-group,n=8):右前外侧第三肋间开胸,经右心房表面送入扩张器行人工房间隔造口术;再经左前外侧第三肋间开胸,将主肺动脉套带环缩。所有动物正常饲养2个月。然后正中开胸,测量跨肺动脉环缩处的压差;分别抽外周动脉、右心房、肺静脉、左心房血液行血气分析,计算Qp/Qs;抽外周静脉血测量血球压积(HCT)和血红蛋白浓度(HB)。 【结果】.对照组8只均存活,平均体重增加到23.0kg。肺血减少组存活6只,平均体重增加到20.6kg。肺血减少组的动脉血氧分压(PaO_2)、动脉血氧饱和度(SaO_2)、HCT和HB分别为52.9 mmHg、85.6%、49.8%和16.6g/dl。对照组的PaO_2、SaO_2、HCT和HB则分别为118.0 mmHg、98.0%、37.9%和12.2g/dl。二组间对应指标相比均有显著性差异(P＜0.001)。利用四点血气值和公式计算所得,肺血减少组的Qp/Qs为0.54:1。 【结论】.应用介入和外科相结合的Hybrid方法,通过人工房间隔造口和肺动脉环缩,成功建立了一种新颖的紫绀型肺血少先天性心脏病动物模型,为研究紫绀型肺血少先心病的临床病理和生理提供了一种有用的工具。 第二部分.肺血减少肺动脉低压时肺组织的病理形态观察和分析 【目的】.利用我们已建立的紫绀型肺血少动物模型,对肺血减少时未成熟肺小血管进行病理形态观察和形态半定量分析,研究肺血减少时未成熟肺小血管的发育特点。 【方法】.健康幼猪16只,分为紫绀肺血减少组(D组,n=8)和对照组(C组,n=8)。首先利用第一部分描述的方法建立紫绀型肺血少先心病动物模型。两组动物正常饲养2个月,麻醉和正中开胸,按照动物活检取材步骤,分别取双侧上、中、下肺叶组织各一块,4%的多聚甲醛充分固定后制片,行H-E和弹性蛋白+Van Gieson染色。光镜和透射电镜观察两组动物肺小血管和肺实质的病理形态特征;利用显微镜-微机图像处理系统,形态半定量测量分析肺小动脉平均中膜厚度(MT)及中膜厚度百分比(MT%)、平均中膜面积(MS)及中膜面积百分比(MS%);计数单位面积肺小动脉数目(APSC)和单位面积肺泡数量(MAN);计算同一张切片所有泡内肺动脉(IAPA)中肌型动脉(CMA)、部分肌型动脉(PMA)和无肌型动脉(NMA)的比例。 【结果】.对照组(C组)8只均存活;肺血减少组(D组)存活6只,D组成功建立紫绀型肺血少先心病动物模型。同对照C组动物相比,光镜观察下D组肺小动脉管腔普遍扩大,形态不规则,管壁厚薄不均,部分弹力纤维层中断、缺失,肺小动脉中层退化和发育不良,部分肌型动脉和无肌型动脉的比例较对照组高,而肌型动脉的比例却相对较低(P＜0.001)。D组肺小动脉MT绝对值、MT%及MS、MS%均显著小于C组(均为P＜0.001)。D组单位面积肺小动脉数(APSC)较C组少(P＜0.01),而肺泡数与C组无显著差异(P＞0.05)。电镜观察显示肺血减少组肺小血管内皮细胞胞质肿胀,基底膜变薄;弹力纤维排列不整齐,有的区域萎缩变薄或中断;血管中层平滑肌轻度萎缩;细胞器减少,线粒体空泡化:部分肺泡Ⅱ型上皮细胞增生明显,板层体丰富。 【结论】.对实验性紫绀型肺血少动物模型的肺组织进行病理形态观察和分析发现,肺血流较正常减少和肺动脉低压时肺小血管发育呈现明显不良或退化,同时伴有外周肺小动脉数量减少,但是对肺泡的形态发育和数量影响不大。形态结构异常影响器官的正常功能,说明保持正常的肺血流或尽早改善肺血少状态是促进外周肺血管正常发育和改善全身状况的关键。 第三部分.肺血少肺动脉低压时肺组织形态变化的生物学基础 【目的】.紫绀型肺血少先心病动物模型的肺血管和肺组织形态结构与正常不同,即发生了细胞外基质重塑。通过测量肺细胞外基质中部分结构性蛋白和细胞因子的变化,分析肺血减少时肺细胞外基质重塑和形态结构变化的生物学基础。 【方法】.健康幼猪16只,分为紫绀肺血减少组(D组,n=8)和对照组(C组,n=8)。首先建立紫绀型肺血少先心病动物模型。两组动物正常饲养2个月,麻醉和正中开胸,均取双侧肺组织,液氮冷冻保存。采用酶联免疫反应方法(Elisa),测量两组肺组织中的基质金属蛋白酶(MMP-2、MMP-9)及基质金属蛋白酶抑制剂(TIMP-1),肺组织Ⅰ型和Ⅲ型胶原纤维(CoⅠ和CoⅢ)和肺血管内皮细胞生长因子(VEGF)含量的变化。 【结果】.对照组(C组)8只均存活;肺血减少组(D组)存活6只,成功建立紫绀型肺血少先心病动物模型。酶联免疫吸附反应(Elisa)测量D组和C组肺组织中基质金属蛋白酶MMP-2的含量分别为56.6±20.3 ng/ml和77.6±20.7 ng/ml,二者有显著性差异(P＜0.01),D组和C组肺组织中基质金属蛋白酶MMP-9的含量分别为4.7±1.0 ng/ml和5.8±1.0 ng/ml,二者有显著性差异(P＜0.01);基质金属蛋白酶抑制剂(TIMP-1)在D组和C组的含量分别为14.5±3.4ng/ml和20.2±3.8ng/ml,二者有显著性差异(P＜0.001)。D组和C组肺组织Ⅰ型(CoⅠ)胶原纤维含量分别为814.6±2006.8 ng/ml和3122.1±2865.1ng/ml,二者有显著性差异(P＜0.01),D组和C组肺血管Ⅲ型(CoⅢ)胶原纤维含量分别为12.7±4.7 ng/ml和8.2±4.2 ng/ml,二者有显著性差别(P＜0.01)。肺血管内皮细胞生长因子(VEGF)在D组和C组的含量分别为13.6±3.0 ng/ml和18.8±3.5ng/ml,二者有显著性差异(P＜0.001)。 【结论】.紫绀型肺血少型先心病动物肺细胞外基质发生构形重建,肺小血管发育不良或退化。同多流量型肺动脉高压的机制相似,肺血减少时肺内环境改变启动各种调控因素,引起细胞外基质中结构性蛋白和细胞因子等成分变化是基质重塑的基础。
[Abstract]:The first part. Interventional and surgical techniques were combined to establish the pulmonary blood disease in young pigs with the animal model
[Objective]. Hybrid adopts a novel surgical intervention and the combination of the establishment of animal model of heart disease with pulmonary blood of young pioneers.
[method] using after birth from 1 to February in young pigs, weighing 8 ~ 12kg, divided into two groups, 8 rats in each group.A) normal control group (C-group, n=8): sham operation group, open chest surgery treatment; B group (D-group) with decreased pulmonary blood flow, n=8): right front third lateral intercostal thoracotomy through the right atrial surface into the dilator of artificial atrial septostomy; the left anterolateral third intercostal thoracotomy, the main pulmonary artery band set shrinkage. All animal normal feeding for 2 months. Then a median sternotomy, measuring cross pulmonary artery pressure; were smoking outside peripheral artery, right atrium and pulmonary vein, analysis, calculation of Qp/Qs of left atrial blood for blood gas drainage; peripheral venous blood measurement of hematocrit (HCT) and hemoglobin concentration (HB).
[results]. 8 rats in control group were alive, the average weight increased to 23.0kg. decreased pulmonary blood group survival was 6, the average weight increased to 20.6kg. decreased pulmonary blood flow and arterial oxygen partial pressure group (PaO_2), arterial oxygen saturation (SaO_2), HCT and HB were 52.9 mmHg, 85.6% 16.6g/dl., and 49.8% in control group PaO_2, SaO_2, HCT and HB were 118 mmHg, 98% 12.2g/dl., 37.9% and two groups of corresponding index showed significant difference (P < 0.001). By four points and blood gas values calculated, reduce pulmonary blood group Qp/Qs 0.54:1.
[Conclusion]. By using the Hybrid method of interventional and surgical combination, through artificial atrial septostomy and pulmonary artery banding, successfully established a novel pulmonary blood less cyanotic congenital heart disease animal model for the study of the pulmonary blood, cyanotic heart disease in clinical pathology and physiology provides a useful tool.
The second part. The pathological observation and analysis of lung tissue when pulmonary blood is reduced to pulmonary arterial pressure
[Objective]. We have established the animal model with cyanotic pulmonary blood, the semi quantitative analysis of morphological and pathological morphology of immature small pulmonary vessels were decreased pulmonary blood flow, developmental characteristics of immature pulmonary small vessels to reduce pulmonary blood.
[method]. 16 healthy piglets were divided into cyanosis group (group D, n=8) and control group (group C, n=8). First establish the animal model of pulmonary blood of the heart with the first part description methods. Two groups of normal animal feed for 2 months, anesthesia and thoracotomy. According to the animal biopsy steps were taken in the lower lobe, bilateral, organization of each one, 4% paraformaldehyde fixed after production, H-E and elastin +Van Gieson staining. The pathological morphological characteristics were observed in the two groups of animal pulmonary small vessels and lung parenchyma by using light microscopy and transmission electron microscopy; microscope image processing system, semi quantitative morphological analysis of pulmonary arterioles in the average film thickness (MT) and intima-media thickness (MT%), the average percentage of membrane area (MS) and membrane area percentage (MS%); the number of counts per unit area of pulmonary artery (APSC) and the number of alveoli per unit area (MAN) with a calculation; cut The proportion of the muscle type artery (CMA), the partial muscle artery (PMA) and the non muscle artery (NMA) in all the alveolar pulmonary artery (IAPA).
銆愮粨鏋溿，

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