自发性高血压大鼠颅内动脉瘤瘤壁血管平滑肌细胞增殖活性的研究

Research of vascular smooth muscle cell proliferation activity of intracranial aneurysms in spontaneously hypertensive rats

目的探讨自发性高血压大鼠（SHR）颅内动脉瘤形成过程中瘤壁血管平滑肌细胞（VSMC）的增殖活性。方法将180只成年雄性大鼠按照随机数字表法均分为对照组与5个实验组（包括1个月组、2个月组、3个月组、4个月组以及5个月组）。实验组采用经背部入路双侧肾动脉后支结扎和左侧颈总动脉结扎建立SHR颅内动脉瘤模型。测定各组大鼠的尾动脉血压，行磁共振血管成像检查观察大鼠的颅内动脉瘤形成情况。取动脉瘤标本，采用HE染色观察动脉瘤的形态学改变，免疫组化方法检测瘤壁增殖细胞核抗原（PCNA）的表达，TUNEL染色测定瘤壁VSMC的凋亡情况。以Westernblot方法检测瘤壁d平滑肌肌动蛋白（α-SMA）、β微管蛋白以及骨桥蛋白（OPN）的表达，通过实时定量PCR法检测瘤壁SM220t和高血压相关基因（HRG-1）mRNA的表达。结果1个月组、2个月组、3个月组、4个月组、5个月组血压逐渐升高，且均高于术前和对照组的血压（均P〈0．05）。4个月组和5个月组可见动脉瘤形成。HE染色可见SHR颅内动脉内膜破坏、消失，中膜层VSMC数目减少，血管壁变薄。与对照组相比，各实验组的动脉壁组织中的PCNA阳性细胞率均明显增高（均P〈0．05），其中4个月组最高，5个月组有所下降。实验组颅内动脉壁VSMC的凋亡率自1个月组至5个月组逐渐升高，且均明显高于对照组（均P〈0．05）。实验组颅内动脉壁α-SMA和β微管蛋白的表达自1个月组至5个月组逐渐下降，且均明显低于对照组（均P〈0．05）；与对照组相比，各实验组OPN的表达均明显增高（均P〈0．05），其中4个月组最高，5个月组则开始下降。实验组颅内动脉壁VSMC的SM22α和HRG-1的mRNA自1个月组至5个月组逐渐下降，且均明显低于对照组（均P〈0．05）。结论SHR颅内动脉瘤形成过程中瘤壁VSMC具有增殖活性，且颅内动脉瘤的形成与瘤壁VSMC的增殖活性减弱、VSMC数量减少密切相关。

Objective To investigate the proliferative activity of vascular smooth muscle cell （VSMC） in the formation of intraeranial aneurysm （IA） in spontaneously hypertensive rats （SHR）. Methods A total of 180 adult male rats were randomly divided into 6 groups including the control, 1-, 2-, 3-, 4- and 5-month experiment groups （n = 30 in each group）. In the 5 experiment groups, the left common carotid artery and posterior branches of both renal arteries of rats were ligated to establish the rat model of spontaneous hypertensive intracranial aneurysms. The arterial blood pressure of the rats in each group was measured, and intracranial arteries were examined with MRA to observe the formation of IAs in rats. The specimens of aneurysms were obtained and morphological changes of IA were observed by HE staining. The expression of proliferating cell nuclear antigen （PCNA） of IA wall in each group was explored with immunohistoehemistry. Apoptosis of IA wall VSMC was detected with the method of TUNEL. The protein levels of α-smooth muscle aetin （α-SMA）, β-tubulin and osteopontin （OPN） in IA wall were detected by Western blot. The mRNA relative levels of SM22α and hypertension-related gene （ HRG-1 ） were tested with quantitative real-time PCR. Results Compared with controls, in experimental groups, the blood pressureof SHR was significantly increased after operation （ all P 〈 0.05 ）. Intracranial aneurysms could be observed in 4 - month and 5 - month experiment groups. The expressions of PCNA were significantly increased, reached the highest levels in the 4-month group, and began to decrease in the 5-month group. The percentage of apoptosis of VSMC of aneurysm wall in the experimental groups was increased significantly. In experimental groups, levels of α-SMA and β-tubulin expression were significantly decreased（ all P 〈 0.05 ）. The expression levels of OPN were increased gradually, reached the highest levels in the 4-month group, and began to decrease in the 5-month group. SM22α and HRG-1 mRNA expressions were gradually decreased （ all P 〈 0.05 ）. Conclusions In the process of IA formation, the aneurysm wall VSMCs demonstrate proliferation activity; and the formation of IA seems to be closely related to the weakening of proliferation activity and number decrease of aneurysmal wall VSMCs.