颅内动脉瘤动物模型的建立及发病机制的研究

发布时间：2018-06-24 03:43

本文选题：颅内动脉瘤 + 动物模型　；参考：《苏州大学》2012年博士论文

【摘要】：第一部分颅内动脉瘤模型制作的研究目的：探寻建立稳定、快捷、理想的颅内动脉瘤动物模型的方法，同时进一步证明颅内动脉瘤的发生、生长、塑形机制。方法：选取48只新西兰大白兔，随机分为A、B、C、D、E五组，A组:新西兰大白兔8只，结扎对侧颈总动脉+2%盐水喂养；B组:兔8只，分叉内膜损伤法+A组法；C组:兔10只，胰弹力蛋白酶浸泡法+A组法；D组:兔12只，分叉内膜损伤法+胰弹力蛋白酶浸泡法+A组法；E组:兔10只，分叉内膜损伤法+胰弹力蛋白酶浸泡法。检测A、D、E三组术前、术后、术后4周的血压；各组于术后4周行CTA颈部血管造影，然后活体解剖左颈总动脉分叉造瘤部位，记录有无成瘤情况，并测量瘤体大小；处死动物，切取瘤体标本，行病理学HE染色，光镜下观察病理组织学变化。结果：A、D、E三组血压变化情况：A、D组术后和术后4周与术前相比P0.0l，有显著性差异；E组术后和术后4周与术前相比P0.0l，无显著性差异；A、D组术后和术后4周与E组术后和术后4周相比P0.0l，有显著性差异。各组成瘤率比较：C、D、E组间p0.05无显著性差异;C、D、E组与A、B组均p0.05有显著性差异。形成动脉瘤体积的比较：D组与E组高度、宽度相比P0.05有显著性差异,长度相比P0.05无显著性差异;C组与D组长度、高度、宽度相比均P0.05有显著性差异。病理组织学变化:正常动脉壁由内膜、中膜和外膜构成，，各层结构保持完整。动脉瘤壁上没有内膜，结构排列紊乱，瘤壁变薄或厚薄不一，有炎性细胞浸润。结论:颈总动脉分叉内膜损伤法+胰弹力蛋白酶浸泡+血流动力因素诱导法成瘤率相对偏高，死亡率低，并可获得体积、病理形态学与人类颅内动脉瘤极为相似的动脉瘤模型，操作简单，损伤小，成瘤周期短，性能稳定可靠，通畅率好，制作费用低，可作为临床前期实验治疗脑动脉瘤和栓塞材料研究的良好模具。同时也证实了颅内动脉瘤的发生、生长塑形及破裂是多种因素综合作用的结果。第二部分PDGF-b、c-Jun、Caspase-3在人脑动脉瘤，兔动脉瘤模型中的表达及意义目的：探讨PDGF-b、c-Jun、Caspase-3在人脑动脉瘤、兔动脉瘤模型中的表达情况，揭示其在脑动脉瘤形成中的潜在作用机制。方法：采用免疫组织化学方法中的Envision法对22例人颅内动脉瘤、8例新西兰大白兔动脉瘤模型、6例正常脑动脉和6例正常兔颈总动脉的石蜡切片标本进行检测PDGF-b、c-Jun、Caspase-3的表达。用免疫组化laserpix图像分析系统进行图像分析，获取PDGF-b、c-Jun、Caspase-3表达的平均光密度值(mean density)进行统计学分析。结果：在22例人颅内囊状动脉瘤中，PDGF-b的表达：强染色6例，明显染色10例，轻度染色4例，无染色2例。正常脑动脉无染色。平均光密度值0.272。c-Jun的表达：强染色12例，明显染色7例，轻度染色2例，无染色1例。正常脑动脉2例轻度染色。平均光密度值0.437。Caspase-3的表达：强染色7例，明显染色9例，轻度染色4例，无染色2例。正常脑动脉无染色。平均光密度值0.284。统计学分析显示：PDGF-b、c-Jun、Caspase-3与对照组比较P0.01；PDGF-b与c-Jun的相关分析:r=0.751，两者表达呈正相关；c-Jun与Caspase-3的相关分析:r=0.812，两者表达呈正相关。此类兔动脉瘤模型标本PDGF-b、c-Jun、Caspase-3较对照组均显示较强表达，对照组均阴性表达。结论：PDGF-b、c-Jun、Caspase-3参与脑动脉瘤的发生、生长、破裂演变过程，脑动脉瘤的形成是多因素综合作用的结果，各因素间存在相互影响、相互关联、相互作用。核转录因子c-Jun可能是颅内动脉瘤发生的病理学因素中血管活性生长因子、凋亡、酶学系列变化、炎性反因等损伤机制中的共同中间环节。为临床开发阻断颅内动脉瘤发生、破裂的新靶点抑制剂提供理论依据。第三部分流体切应力对动脉内皮细胞PDGF-b、c-Jun、Caspase-3mRNA表达的影响和意义目的:探讨高流体切应力作用下动脉内皮细胞PDGF-b、c-Jun、Caspase-3mRNA的表达水平和规律,揭示血流动力学因素在颅内动脉瘤形成演变过程中的具体分子病理学机制. 方法:选择人胸主动脉血管内皮细胞培养、传代作为内皮细胞功能研究的细胞源，应用体外高切应力流场加载机,分别将各组内皮细胞置于切应力加载机的流室腔槽内。a.施加高强度流体切应力(40dyne/cm2)分别作用0h、1h、4h、6h,然后应用RT-PCR技术检测不同作用时间点内皮细胞PDGF-b、c-Jun、Caspase-3的转录水平和规律；b.施加正常强度流体切应力(20dyne/cm2)4h仍应用RT-PCR技术检测内皮细胞PDGF-b、c-Jun、Caspase-3的转录水平；c.比较同时间点（4h）高强度流体切应力与正常强度流体切应力PDGF-b、c-Jun、Caspase-3的转录水平的差异性。结果：高切应力作用下，PDGF-b组：1小时组内皮细胞PDGF-b转录水平就显著升高，4小时、6小时较1小时组有回落，且存在统计学差异，但仍较未加载切应力(0h)有显著差异，而6小时后又有回升趋势;c-Jun组：未加载切应力的内皮细胞组c-Jun mRNA表达几乎为零，而在1h、4h、6h时间点的高切应力加载组渐进性、与时间呈正相关性升高，与未加载切应力组比较有显著差异；随着切应力作用时间的延长，6h、4h、1h各组比较均有显著性差异;Caspase-3组：未加载流场的对照组(0h组)内皮细胞也检测到相对中等量的Caspase-3mRNA表达，而在40dyne/cm2的高切应力作用下，1h组表达量明显升高，较对照组(0h组)有显著性差异；但在4h组下降到对照组水平以下，较对照组(0h组)亦有显著性差异；6h组有回升趋势，升高到对照组(0h组)水平以上，仍接近对照组水平，比较无统计学差异。1h组较4h组、6h组有显著性差异。同时间点（4h）不同强度切应力比较：无流体切应力、正常强度流体切应力、高强度流体切应力PDGF-b、c-Jun的转录水平的差异有显著性p0.05;4h时点Caspase-3组随着切应力增强渐下降且p0.05有显著性差异。结论：高强度的流体切应力可诱导人胸主动脉内皮细胞内PDGF-b mRNA、c-JunmRNA、Caspase-3mRNA的转录表达。而且随着作用时间延长1h、4h、6h时间点各有其特征性表达规律。结合本文第二部分结果可推测出高流体切应力可能通过调节PDGF-b、c-Jun、Caspase-3的表达水平，进而诱导颅内动脉瘤的发生、形成塑形和破裂。在我们的实验研究中Caspase-3的表达水平在无流体切应力、正常强度流体切应力、高强度流体切应力4h时点随着切应力增强渐下降且有显著性差异，提示我们需进行更多更长时点进一步深入的研究，来揭示其作用表达规律。
[Abstract]:The study of the first part of the model of intracranial aneurysm
Objective: To explore a stable, quick and ideal animal model of intracranial aneurysms, and further prove the occurrence, growth and shaping mechanism of intracranial aneurysms.
Methods: 48 New Zealand white rabbits were randomly divided into groups of A, B, C, D, E five, group A: 8 New Zealand white rabbits, 8 rabbits were ligated to the lateral common carotid artery, +2% brine, 8 rabbits in group B, and +A group method of bifurcation intima injury, 10 rabbits, 10 rabbits and trypsin immersion method, 12 rabbits, bifurcation intima injury and trypsin immersion method Group E: group E: Rabbit 10, bifurcation intima injury and trypsin soaking. Test the blood pressure of group A, D, E after operation, postoperative, 4 weeks postoperatively; each group performed CTA neck angiography at 4 weeks after operation, then dissected the part of the left common carotid artery bifurcation, recorded the tumor formation, and measured the size of the tumor; the animals were killed and the tumor body marks were executed. The pathological changes were observed by HE staining and histopathological changes were observed under light microscope.
Results: A, D, E three groups of blood pressure changes: A, D group postoperative and 4 weeks after the operation compared with preoperative P0.0l, there were significant differences; E group postoperative and 4 weeks after the operation compared with preoperative P0.0l, no significant difference; A, D group and 4 weeks after the operation and E group, compared with 4 weeks after the 4 weeks, there were significant differences. The significant difference between C, D, E group and A and B group. The comparison of the volume of the aneurysm: the height of the D group and the E group, the width compared with P0.05, there is no significant difference in the length compared with the P0.05; the C group is significantly different from the length, height and width of the D group. The pathological changes: the normal arterial wall is from the intima, middle The membrane and outer membrane are made up. The structure of each layer is intact. There are no intima on the wall of the aneurysm, the structure is arranged in disorder, the wall of the aneurysm is thin or thick, and there is inflammatory cell infiltration.
Conclusion: the common carotid artery branched intima damage method, pancreatic elastase immersion + hemodynamic factor induction method is relatively high, the mortality is low, the volume is low, the pathomorphology and the human intracranial aneurysm are very similar to the aneurysm model. The operation is simple, the injury is small, the cycle of the tumor is short, the performance is stable, the patency rate is good, the cost of patency is good and the production fee is good. Low use can be used as a good mold for the treatment of cerebral aneurysms and embolic materials in preclinical trials. It has also confirmed the occurrence of intracranial aneurysms, and the growth, shaping and rupture are the results of a variety of factors.
The second part is the expression and significance of PDGF-b, c-Jun and Caspase-3 in human cerebral aneurysms and rabbit aneurysm models.
Objective: To investigate the expression of PDGF-b, c-Jun and Caspase-3 in human cerebral aneurysms and rabbit aneurysm models, and to reveal their potential mechanisms in the formation of cerebral aneurysms.
Methods: the expression of PDGF-b, c-Jun and Caspase-3 in 22 human intracranial aneurysms, 8 New Zealand white rabbit aneurysm models, 6 normal cerebral arteries and 6 normal rabbits' common carotid artery were detected by Envision method, and the image analysis was obtained by immunohistochemical laserpix image analysis system. The average optical density (mean density) expressed by PDGF-b, c-Jun and Caspase-3 was analyzed statistically.
Results: in 22 cases of human intracranial saccular aneurysms, the expression of PDGF-b: strong staining in 6 cases, obvious staining in 10 cases, mild staining in 4 cases, without staining in 2 cases, normal cerebral artery without staining. The expression of mean light density value 0.272.c-Jun: strong staining 12 cases, obvious dyeing 7 cases, mild staining 2 cases, no dyeing 1 cases. 2 mild coloring and average light density of normal cerebral artery. The expression of degree value 0.437.Caspase-3: strong staining in 7 cases, obvious staining in 9 cases, mild staining in 4 cases, non staining in 2 cases and normal cerebral artery without staining. The mean light density value 0.284. statistical analysis showed that PDGF-b, c-Jun, Caspase-3 were compared with the control group P0.01; r=0.751, the correlation between PDGF-b and c-Jun was positive correlation; c-Jun and Caspase-3. The correlation analysis: r=0.812, the expression of the two was positive correlation. The rabbit aneurysm model specimens PDGF-b, c-Jun, Caspase-3 were more expressed than the control group, and the control group were all negative.
Conclusion: PDGF-b, c-Jun and Caspase-3 are involved in the occurrence, growth, and rupture process of cerebral aneurysm. The formation of cerebral aneurysms is the result of multiple factors. There are interrelated, interrelated and interrelated factors between the various factors. The nuclear factor c-Jun may be a vasoactive growth factor and apoptosis in the pathological factors of intracranial aneurysm. It provides a theoretical basis for the development of new target inhibitors for the development of intracranial aneurysms and rupture of intracranial aneurysms.
The third part is the effect of fluid shear stress on the expression of PDGF-b, c-Jun and Caspase-3mRNA in arterial endothelial cells.
Objective: To explore the expression level and regularity of PDGF-b, c-Jun and Caspase-3mRNA in arterial endothelial cells under high fluid shear stress, and to reveal the specific molecular pathological mechanism of hemodynamic factors in the process of the formation and evolution of intracranial aneurysms.
Methods: the endothelial cell culture of human thoracic aorta was selected as the source of endothelial cell function study, and the high shear stress field loading machine was applied in vitro. The high intensity fluid shear stress (40dyne/cm2) was applied to.A., 1H, 4h, 6h respectively in the flow chamber slot of the shear stress loader. Then, RT-PCR was applied to RT-PCR, respectively, and then RT-PCR was applied to RT-PCR, respectively. The transcriptional level and regularity of PDGF-b, c-Jun, Caspase-3 of endothelial cells at different time points were measured, and B. applied to normal intensity fluid shear stress (20dyne/cm2) 4H to detect the transcription level of PDGF-b, c-Jun, Caspase-3 of endothelial cells, and C. compare the shear stress of high intensity fluid with normal intensity fluid at the same time point (4h). The differences in transcriptional level of PDGF-b, c-Jun and Caspase-3.
Results: under high shear stress, group PDGF-b: the PDGF-b transcriptional level of endothelial cells in the 1 hour group increased significantly, 4 hours and 6 hours compared with the 1 hours group, and there was a statistical difference, but there was a significant difference compared with the unloaded shear stress (0h), but there was a rebound trend after 6 hours; group c-Jun: c-Jun mRNA of the endothelial cell group that did not load the shear stress. The expression was almost zero, while the progressive stress loading group in 1H, 4H and 6h time points was progressive, with a positive correlation with time, and significant difference compared with the unloaded shear stress group. With the prolongation of the shear stress action time, there were significant differences in 6h, 4H and 1H groups; Caspase-3 group: the control group (0h group) of the unloaded flow field was also endothelial cells. The expression of relatively medium Caspase-3mRNA was detected, and the expression of 1H group increased significantly under the high shear stress of 40dyne/cm2, compared with the control group (0h group), but in 4H group decreased to the control group, compared with the control group (0h group), there was a significant difference between the control group and the control group (Group 0h); the 6h group had a rising trend and increased to the level of the control group (0h group). There was no significant difference between the.1h group and the 4H group. There was significant difference between the 6h group and the control group.
The difference of shear stress at the same time point (4h): no fluid shear stress, normal strength fluid shear stress, high intensity fluid shear stress PDGF-b, c-Jun transcriptional level difference is significant P0.05; 4H time point Caspase-3 group gradually decreases with shear stress and P0.05 has significant difference.
Conclusion: high intensity fluid shear stress can induce the transcriptional expression of PDGF-b mRNA, c-JunmRNA, Caspase-3mRNA in human thoracic aorta endothelial cells. Moreover, with the time of action prolonging 1H, 4h, 6h point, there are characteristic expression rules. Combined with the results of the second part of this article, we can speculate that the high fluid shear stress may be regulated by PDGF-b, c-Jun, Casp. The expression level of ase-3, which leads to the occurrence of intracranial aneurysms, leads to formation and rupture. In our experimental study, the expression level of Caspase-3 is in the absence of fluid shear stress, normal strength fluid shear stress, and high intensity fluid shear stress 4H time points with the shear stress increasing gradually decreasing and significant difference, suggesting that we need to carry on more longer. Further study is carried out to reveal its function and expression pattern.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R743;R-332

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