大鼠颅内动脉瘤壁平滑肌细胞增殖活性探讨的实验研究

发布时间：2018-06-22 03:33

本文选题：颅内动脉瘤 + 血管平滑肌细胞　；参考：《苏州大学》2016年硕士论文

【摘要】：目的:颅内动脉瘤(intracranial aneurysm,IA)具有自发性破裂出血的倾向,致残率和致死率都很高。目前,在临床上关于IA的治疗仅仅局限于手术和介入治疗,而对动脉瘤壁进行修复等非手术治疗几乎没有。在对IA发病机制的研究中,我们发现动脉瘤壁血管平滑肌细胞(vascular smooth muscle cell,VSMC)具有增殖活性功能,结合目前生物治疗和基因治疗的发展,我们萌生了为未破裂颅内动脉瘤壁进行修复治疗的设想。VSMC是动脉壁组成的主要部分,在IA形成过程中,可表现出很高的增殖活性与可塑性,可见IA形成过程中血管壁VSMC的增殖将成为修复研究的主要方向。因此,本实验通过建立大鼠颅内动脉瘤模型对大鼠颅内动脉瘤壁VSMC增殖活性进行探讨,目的在于增强动脉瘤壁VSMC的增殖活性,增加平滑肌层,为今后人颅内动脉瘤壁的修复治疗提供理论基础。方法:成年雄性自发性高血压大鼠(spontaneously hypertensive rat,SHR)180只,随机分为6组:对照组和实验1、2、3、4、5月组(每组30只)。实验组采用经背部入路行双侧肾动脉后支结扎和左侧颈总动脉结扎,对照组仅暴露血管,不结扎。各组术后1周开始喂养含1%氯化钠的饮用水和0.12%β-氨基丙腈饲料。每组大鼠术前及处死前测量室温安静状态下尾动脉血压,处死前行7.0MRA检查大鼠颅内动脉。取下动脉瘤标本,用HE染色观察动脉瘤的病理变化,免疫组化观察瘤壁增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达和免疫荧光染色和蛋白质印迹(Westernblot,WB)观察瘤壁α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、β-微管蛋白(β-Tubulin)和骨桥蛋白(osteopontin,OPN)的表达以及实时定量PCR观测瘤壁SM22α与高血压相关基因(hypertension-related gene,HRG-1)mRNA的表达来了解动脉瘤壁VSMC的增殖,末端标记法(terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling,TUNEL)染色测定瘤壁VSMC的凋亡。结果:1.血压变化:对照组平均血压缓慢升高。实验组平均血压从1月组起至5月组逐渐升高,均明显高于术前和对照组(P0.05)。2.MRA检测情况:每组随机抽取6只SHR行7.0 MRA检测。对照组颅内动脉左右对称、粗细均匀,无明显扩张增粗。实验组所有检测的右侧颅内动脉均有不同程度的增粗,血管病变率达100%,其中,4月组有3/6在增粗的血管上可见动脉瘤形成,5月组有5/6可见动脉瘤形成。3.动脉瘤病理变化:对照组颅内动脉无明显破坏,各层结构保持完整。实验组颅内动脉从1月组起内膜层开始破坏、VSMC退变、VSMC数目及层数减少、动脉壁逐渐变薄,至4、5月组时内膜层已消失、弹性纤维断裂,并有炎性细胞浸润,其中,5月组最明显,动脉壁最薄。4.免疫组化结果:对照组PCNA阳性细胞率低下。实验组PCNA阳性细胞率从1月组起至5月组与对照组相比均明显增高(P0.05);其中,4月组最高,5月组开始下降,与4月组相比有显著差异(P0.05)。5.Western blot结果:对照组α-SMA和β-tubulin呈高表达,而OPN几乎不表达。实验组α-SMA和β-tubulin的表达从1月组起至5月组逐渐下降,均明显低于对照组(P0.05);而OPN的表达从1月组起至5月组与对照组相比均明显增高(P0.05);其中,4月组最高,5月组开始下降,与4月组相比差异显著(P0.05)。6.免疫荧光结果:对照组α-SMA和β-tubulin呈高表达,而OPN几乎不表达。实验组α-SMA和β-Tubulin的相对荧光强度从1月组起至5月组逐渐降低,均明显低于对照组(P0.05);而OPN的相对荧光强度从1月组起至5月组与对照组相比均明显增高(P0.05),其中,4月组最高,5月组开始下降,与4月组相比差异显著(P0.05)。7.实时定量PCR结果:对照组SM22α和HRG-1 mRNA相对表达较高。实验组SM22α和HRG-1 mRNA相对表达从1月组起至5月组逐渐下降,均明显低于对照组(P0.05)。8.TUNEL染色结果:对照组VSMC凋亡率低下。实验组VSMC凋亡率从1月组起至5月组逐渐升高,均明显高于对照组(P0.05)。结论:实验性大鼠IA形成过程中动脉瘤壁VSMC具有增殖活性功能,且具有明显的增殖活跃期;IA的形成与动脉瘤壁VSMC的增殖活性相对减弱密切相关。
[Abstract]:Objective: intracranial aneurysm (IA) has a tendency to spontaneous rupture and hemorrhage, and the rate of disability and lethality are high. Currently, the clinical treatment of IA is limited to surgery and interventional therapy, but nonsurgical treatment for the repair of the aneurysm wall is few. In the study of the pathogenesis of IA, we found the movement. Vascular smooth muscle cell (VSMC) has the function of proliferative activity. Combined with the development of biological therapy and gene therapy, we conceive that.VSMC is the main part of the arterial wall composition, which can show high proliferation in the process of IA formation. The proliferation of the vascular wall VSMC in the formation of IA will be the main direction of the repair research. Therefore, the rat intracranial aneurysm model was established to explore the VSMC proliferation activity of the intracranial aneurysm wall in rats. The aim of this experiment is to enhance the proliferation activity of the aneurysm wall VSMC and increase the smooth muscle layer for the future human intracranial. The repair of aneurysm wall provides a theoretical basis. Methods: 180 adult male spontaneously hypertensive rats (spontaneously hypertensive rat, SHR) were randomly divided into 6 groups: the control group and the experimental 1,2,3,4,5 month group (30 rats in each group). The experimental group was ligation of the posterior branches of the bilateral renal artery and the left common carotid artery by the back approach, and the control group was only violent. 1 weeks after operation, 1% sodium chloride containing drinking water and 0.12% beta aminonitrile feed were fed. The blood pressure of the caudal artery was measured at room temperature and quiet before and before the operation. Before and before the operation, the intracranial arteries of the rats were examined by 7.0MRA. The specimens of the aneurysm were taken and the pathological changes of the aneurysm were observed by HE staining and the immunohistochemical view was observed. The expression of proliferating cell nuclear antigen (PCNA) and immunofluorescence staining and Western blot (Westernblot, WB) observation of the tumor wall alpha smooth muscle actin (alpha -smooth muscle actin, alpha -SMA), the expression of beta microtubulin (beta -Tubulin) and osteopontin and real-time quantitative observation The tumor wall SM22 alpha and hypertension-related gene (HRG-1) mRNA were expressed to understand the proliferation of the aneurysm wall VSMC. Terminal labeling (terminal deoxynucleotidyl transferase-mediated dUTP nick end) staining was used to determine the apoptosis of the tumor wall. Results: 1. blood pressure changes: the mean blood pressure in the control group increased slowly. The average blood pressure in the experimental group increased gradually from the January group to the May group, which was significantly higher than that in the pre operation and the control group (P0.05).2.MRA detection: each group was randomly selected 6 SHR for 7 MRA tests. The control group was symmetrical and thin, without obvious dilation and thickening. All of the right intracranial arteries in the experimental group were thickened in varying degrees. The rate of vascular lesions was 100%, of which, in the April group, the aneurysm was visible on the thickened vessels, and the pathological changes of the aneurysm formed in the group of 5/6 were seen in the May group. There was no obvious destruction of the intracranial artery in the control group, and the structure of each layer remained intact. The intracranial artery in the experimental group began to destroy from the inner membrane of the January group, the VSMC degeneration, the number of VSMC and the number of layers decreased in the experimental group. The arterial wall gradually thinned, the intima layer disappeared, the elastic fibers broke and the inflammatory cells infiltrated in the 4,5 month group. Among them, the May group was the most obvious, the thinnest.4. immunohistochemical results were found in the control group, and the rate of PCNA positive cells in the control group was low. The rate of PCNA positive cells in the experimental group increased significantly from the January group to the May group (P0.05), and 4 The month group was the highest, and the May group began to decline. Compared with the April group, there was a significant difference (P0.05).5.Western blot results: the control group of alpha -SMA and beta -tubulin were highly expressed, and OPN almost did not express. The expression of alpha -SMA and beta -tubulin in the experimental group decreased gradually from the January group to the May group, all significantly lower than the control group (P0.05), while the expression of OPN was from January to May group. Compared with the control group (P0.05), the April group was the highest and the May group began to decline. Compared with the April group, the difference was significant (P0.05).6. immunofluorescence results: the control group of alpha -SMA and beta -tubulin were highly expressed, and the OPN almost did not express. The relative fluorescence intensity of the alpha -SMA and beta -Tubulin in the experimental group decreased gradually from the January group to the May group, both significantly lower. Compared with the control group (P0.05), the relative fluorescence intensity of OPN increased significantly from the January group to the May group (P0.05), which was the highest in the April group, and the May group began to decline. Compared with the April group, the difference was significant (P0.05).7. real-time quantitative PCR results: the relative expression of SM22 alpha and HRG-1 mRNA in the control group was higher. The SM22 A and HRG-1 mRNA in the experimental group were relatively expressed. From the January group to the May group gradually decreased, both significantly lower than the control group (P0.05).8.TUNEL staining results: the control group VSMC apoptosis rate is low. The apoptosis rate of VSMC in the experimental group increased gradually from the January group to the May group, all obviously higher than the control group (P0.05). Conclusion: the arterial wall VSMC has the proliferative activity function in the experimental rat IA formation process, and it has a bright future. The proliferative phase was significantly correlated with the formation of IA and the relative attenuation of VSMC proliferation in aneurysm walls.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R743

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