一种新型弹力蛋白酶诱导兔动脉瘤模型的建立

发布时间：2018-10-04 22:25

【摘要】：实验背景:颅内动脉瘤是由于动脉血管壁局部较脆弱、内部压力增高，导致局部血管异常而引起的脑血管瘤样突起，发生率为0.2%一1%。由于动脉瘤破裂的不确定性以及破裂后的高致残率和致死率，动脉瘤始终是神经外科需要研究的重点疾病之一。为探讨动脉瘤的形态学、病理学、演变过程，我们需要建立可信可靠的动脉瘤模型进行血流动力学、病理生理学以及临床治疗等多方面研究。目前在理想状态下制作出的动脉瘤动物模型应具备下列特点:(1)无论从形态学特征或是从病理学特征都与真正的动脉瘤相同或近似;(2)形态结构应相对具有稳定性，并且应具有较好的可重复性;(3)制作动脉瘤模型的方法应简便易行，操作所需时间短，所需费用较低;(4)动脉瘤需要有适当的体积，有利于实验观察及进一步操作;(5)动脉瘤与载瘤动脉在实验期间不发生血栓，动脉瘤壁有一定强度，不易破裂。目前所建立的与人类形态较相似的动脉瘤动物模型主要有鼠、兔、犬、猪、羊和猴等动物。因为现有的通过各种实验方法建立起来的任何一种动物模型还无法完全模拟人类动脉瘤的生物学特性，所以需要我们继续努力研究出更加理想更加合适的动脉瘤模型。因此，理想的动脉瘤动物模型的发展方向应趋向于制作简单、可重复性高;用时短，成本低;并且血流动力学、组织学以及病理学等与人类动脉瘤更加接近。本实验应用胰弹力蛋白酶建立兔囊状动脉瘤模型，并通过股动脉造影及病理学检查检测模型。实验材料与方法:(1)术前准备:取成年新西兰大白兔12只(体重2 .4一2.skg)，雌雄不限，分为实验组9只和对照组3只。麻醉应用陆眠灵0.2 ml/1(g，戊巴比妥钠巧mg，，kg，青霉素10万单位瓜g加入生理盐水中稀释，分别肌肉注射。(2)注入胰弹力蛋白酶:应用外科手术解剖分离右侧颈外动脉，在颈外动脉下方垫乳胶片并穿两条真丝编线于其后，结扎颈外动脉远端，颈外动脉起始部以动脉瘤夹临时夹闭，用装有生理盐水的1毫升无菌注射器穿入盲端颈外动脉反复冲沈后，将弹力蛋白酶20单位注入到盲端颈外动脉内后结扎，实验组在颈外动脉盲端注入胰弹力蛋白酶，对照组在颈外动脉注入生理盐水。等待20分钟后撤出动脉临时阻断夹，确认无出血后缝合皮肤。(3)术后经股动脉造影复查:分别于术后3天、3周行股动脉造影监测动脉瘤模型。3天后复查在兔右侧股动脉搏动最强处切开皮肤，分离皮下及肌肉组织，小心分离股动脉，远端股动脉结扎，套管针穿刺后，置入短导丝，随后置入5F鞘，推入肝素盐水肝素化。微导管在微导丝辅助下置入主动脉弓造影，随后置入右侧cCA，在右侧颈内动脉开口处推入2ml造影剂，动脉瘤清晰可见。随后给予三维旋转造影。3周后在兔左侧股动脉行血管造影复查，方法同前。(4)病理学检查:给予三维旋转造影后，在静脉注入过量戊巴比妥那处死兔子，分离颈部组织，取下动脉瘤，置入10%EP醛溶液中固定备用。两天后，将标本用PBS缓冲溶液反复冲洗3次，每次冲洗五分钟，再将标本放入甲醛溶液中封闭一周以备用。经过乙醇梯度脱水，包埋、修块、半薄切片制成石蜡切片。再将石蜡切片经HE染色后置于光镜下观察。实验结果:(1)术后3天实验组9只兔子均形成动脉瘤。动脉瘤长径5 .0士1.20二，宽径3.4士0.70二，载瘤动脉直径3_2士0.53二;术后3周动脉瘤长径5 .12士1 .24二，宽径346士0.70二，载瘤动脉直径3一2士0.52二。实验组动脉瘤长径、宽径、载瘤动脉直径于术后3天、3周测量的结果分别比较p值均大于0.05，各观察项目在不同观察时期均无明显差异。对照组3只兔子分别于术后3天和3周未见动脉瘤形成，血管内造影显示右侧颈外动脉残端封闭。(2)实验组术后3周做病理学检查发现H一E染色示动脉瘤顶部新生的内膜形成，瘤壁内膜增生，有炎性细胞侵润，部分可见瘤壁内膜变厚，且瘤顶部可见部分血栓机化。对照组术后3周H一E染色示有血栓形成并将右侧颈外动脉完全封堵，右侧颈外动脉残端内有血栓机化，并可见形成了新生的内膜。实验结论:1.采用胰弹力蛋白酶注射方法成功的建立兔囊状动脉瘤模型。2.通过DSA检测方法证实建立的动脉瘤模型在形态学上与人颅内动脉瘤相似。3.通过本实验建立的兔囊状动脉瘤模型成功率高，实验操作简便、易推广、制作时间短、存活率高。4.通过本实验方法所建立的兔囊状动脉瘤模型形念学及病理学与人类动脉瘤相似，故可用于血管内介入治疗、血流动力学及发病机制方面的研究。
[Abstract]:Background: The incidence of intracranial aneurysm was 0. 2% 1%, due to the local fragility of arterial wall, the increase of internal pressure, and the occurrence of cerebral angioma-like protrusion caused by local vascular abnormality. Aneurysm is always one of the key diseases in neurosurgery, due to the uncertainty of aneurysm rupture and the post-fracture high disability rate and aneurysm rupture. To investigate the morphology, pathology and evolution of aneurysm, we need to establish a credible and reliable model of aneurysm for hemodynamic, pathophysiology and clinical treatment. An animal model of aneurysm produced under ideal conditions should be characterized by (1) the same or similar characteristics as true aneurysms, either from morphological characteristics or from pathological characteristics; (2) morphological structures should be relatively stable and should have better repeatability; (3) The method for manufacturing the aneurysm model is simple and feasible, the time required for operation is short, the required cost is lower, and (4) the aneurysm needs a proper volume, which is beneficial to experimental observation and further operation; and (5) the aneurysm and the tumor-carrying artery do not have thrombus during the experiment, The aneurysm wall has certain strength and is not easy to crack. An animal model of aneurysms similar to human form is currently established with rats, rabbits, dogs, pigs, sheep and monkeys. Because any animal model established by various experimental methods can not fully mimic the biological characteristics of human aneurysms, we need to continue our efforts to develop a more ideal and more suitable aneurysm model. Therefore, the development direction of an ideal aneurysm animal model should tend to be simple, reproducible, short in time, low in cost, and more accessible to human aneurysms, such as hemodynamics, histology, and pathology. In this experiment, the rabbit saccular aneurysm model was established by tryptic protease, and the model was detected by femoral artery angiography and pathology. Materials and Methods: (1) Pre-operative preparation: 12 adult New Zealand white rabbits (weighing 2. 4-2. skg), male and female, were divided into experimental group 9 and control group. Group 3. The anesthesia was applied to 0. 2 ml/ 1 (g, 5 mg/ kg) and 10 000 units of penicillin into normal saline for dilution, respectively. Meat injection: (2) injection of pancreatic elastic protease: apply surgical anatomy to separate right external carotid artery, cushion latex sheet under external carotid artery and wear two silk braided wires, then tie up the distal end of external carotid artery, and the initial part of external carotid artery is clamped on the aneurysm. When a sterile syringe containing physiological saline was inserted into the external carotid artery of the blind end after repeated punching, 20 units of elastic protease were injected into the external carotid artery of the blind end for ligation. The experimental group injected the pancreatic elastic protease into the blind end of the external carotid artery, and the control group was injected into the external carotid artery. Saline. After waiting for 20 minutes, withdraw the temporary occlusion clip of the artery and confirm that there is no post-bleeding seam Three days post-operative femoral artery angiography was performed to monitor the aneurysm model. After 3 days, the skin was dissected on the right femoral artery at the right side of the rabbit, subcutaneous and muscle tissue were separated, and the femoral artery and the distal femoral artery were separated carefully. Vein ligation, puncture of trocar, put into short lead wire, then put into 5F, and push into heparinized saline. heparinized. Microcatheter was inserted into aortic arch with the aid of microguidewire and then placed on the right cCA, and 2ml contrast agent was pushed into the right internal carotid artery opening, and the aneurysm was cleared. 3-dimensional rotational angiography followed by angiography review of the left femoral artery of the rabbit after 3 weeks. (4) Pathology Examination: After three-dimensional rotational angiography, rabbits were sacrificed after intravenous injection of excess pentothal, the neck tissue was separated, the aneurysm was removed, and 10% EP aldehyde solution was placed. after two days, repeatedly washing the specimen with PBS buffer solution for 3 times, washing for five minutes each time, and placing the specimen in a formaldehyde solution for one week preparing stone by using ethanol gradient dehydration, embedding, repairing block and semi-thin slice The paraffin sections were sliced. The paraffin sections were then stained with HE and placed under light microscope. The experimental results were as follows: (1) 9 rabbits in the experimental group after 3 days of operation The aneurysm was formed in the aneurysm. The diameter of the aneurysm was 5. 0 卤 1. 20 di, the wide diameter was 3. 4 卤 0.70II, the diameter of the tumor bearing artery was 3 _ 2 卤 0.053 2. The diameter of the aneurysm was 5. 12 卤 1. 24 2 in 3 weeks after the operation. The width of the aneurysm was 346 卤 0.70II, and the diameter of the tumor-bearing artery was 3. The diameter, width and diameter of the aneurysm in the experimental group were greater than 0. 05 in the 3-week and 3-week period respectively. No significant difference was found in the period. Three rabbits in the control group were not formed in 3 days and 3 weeks after operation, and the right neck was displayed by angiography. (2) The pathological examination of 3 weeks after operation in the experimental group showed that H-E staining showed the formation of neointima in the top of the aneurysm, the intimal hyperplasia of the tumor wall, the invasion and moistening of the inflammatory cells, the thickening of the endometrial wall and the top of the tumor. Partial thrombosis was found in the control group after 3 weeks H-E staining showed thrombus formation and the right external carotid artery was completely blocked, and there was thrombosis in the left and right external carotid artery. It became the neointima. The experiment conclusions: 1. The successful construction of the method of pancreatic elastic protease injection A model of rabbit saccular aneurysm was established. The model of aneurysm established by DSA was found to be in morphology. and 3. The rabbit saccular aneurysm model established by the experiment is simple and convenient to operate, easy to popularize and prepare. Conclusion The model shape and pathology of rabbit saccular aneurysm established by this method are similar to that of human aneurysm, so it can be used for endovascular interventional therapy and hemodynamics.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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