Cx43RNA干扰防治迟发性缺血性神经功能障碍（DIND）的体内实验研究

发布时间：2018-04-16 07:46

本文选题：SAH + 缝隙连接　；参考：《南昌大学》2014年硕士论文

【摘要】：目的：自发性蛛网膜下腔出血（SAH）占脑血管意外的5%左右，在脑血管意外中仅低于脑内出血和脑梗死的发病率，但SAH的病死率明显高于后两者[1]，并且目前缺乏特效治疗措施。SAH后出现的迟发性缺血性神经功能障碍（DIND）是导致高致死高致残率的主要原因。DIND一直是神经外科学者研究的重点和热点。DIND的致病因素，公认的主要的两个理论：即大血管的延迟性痉挛，及微血管的功能紊乱。我们实验室前期研究表明：缝隙连接（GJ）可能在自发性蛛网膜下腔出血后的DIND病理过程中起到十分重要的作用。应用特异性的Cx43siRNA可有效地沉默缝隙连接蛋白43（Cx43）的表达水平，同时能减轻脑血管痉挛的程度[40]。在前期研究的基础上，本实验拟验证：有效下调缝隙连接Cx43后对SAH后发生的DIND的影响。方法： 1、二次注血模型的建立：采用普通级SD大鼠，腹腔麻醉后抽取尾静脉血约0.3ml备用，体式显微镜下枕大池穿刺，见清亮脑脊液流出约0.1ml，将尾静脉血注入，次日进行同前步骤，完成整个造模过程。 2、SAH模型的鉴定：分为单纯SAH造模组和正常组（假手术组），第七天应用墨汁染色法测量基底动脉直径及神经功能评分评价神经功能缺损，鉴定SAH模型。 3、病毒的沉默效率检测：采用腺病毒枕大池注射设立病毒处理组，单纯SAH组和正常组对照。分1d，3d，7d，14d四个时间点处死SD大鼠，取基底动脉，Western bloting检测病毒沉默Cx43的效率。 4、神经功能评分：单纯SAH造模组和正常组，预处理组，处理组等分7d，14d两个时间行神经功能评分（改良的garcia JH和beam balance评分法）。 5、MRI检测：正常组和单纯SAH造模组，预处理组，处理组分7d，14d两个时间点行MRI（DWI，PWI）检测，，包括脑血流量（CBF），表观弥散系数（ADC）值的检测。结果： 1、印度墨汁染色法检测基底动脉直径，造模后7天，单纯SAH造模组的基底动脉直径明显小于假手术对照组。 2、腺病毒沉默效率检测：腺病毒脑池注射可以有效的沉默脑血管的Cx43表达。 3、神经功能评分：改良的garcia评分法：SAH组与正常组比，7d，出现神经功能缺损症状，14天神经功能缺损症状与正常组比无明显差异；所有的实验处理组与单纯SAH组比较，均不存在明显的神经功能缺损；Beam balance评分法，7d，14d所有组别之间不存在明显差异。 4、MRI结果：SAH组与正常组比较，7d出现CBF及ADC值的下降；病毒预处理组和病毒处理组与单纯的SAH造模组比，7d可以改善CBF，14天未能改善CBF，7d，14d均未能改善ADC值；甘珀酸预处理、甘珀酸处理组、病毒溶媒预处理组、病毒溶媒处理组、甘珀酸溶媒预处理组及甘珀酸溶媒处理组与单纯SAH组比较，在7d和14d两个时间点未能改善CBF，ADC值。结论： 1、自体尾静脉血二次枕大池注血模型能够再现SAH的病程。 2、枕大池注射腺病毒，特异性下调Cx43后可以改善SAH后的CBF，提示能够改善SAH的预后。
[Abstract]:Objective:Spontaneous subarachnoid hemorrhage (SAH) accounts for about 5% of cerebrovascular accidents, and is only lower than the incidence of intracerebral hemorrhage and cerebral infarction in cerebrovascular accidents.However, the fatality rate of SAH is significantly higher than that of the latter two [1], and the lack of special therapeutic measures. The delayed ischemic neurological dysfunction (DINDD) is the main cause of high mortality and high disability rate. Dind has always been the research of neurosurgery scholars.Focus and focus on the pathogenic factors of .DIND,Two major theories are generally accepted: delayed vasospasm of large vessels and dysfunction of microvasculature.Our previous laboratory studies have shown that gap junction (GJ) may play an important role in the pathological process of DIND after spontaneous subarachnoid hemorrhage.The application of specific Cx43siRNA can effectively silence the expression of gap junction protein 43 (Cx43) and reduce the degree of cerebral vasospasm [40].On the basis of previous studies, this experiment is intended to verify the effect of down-regulation of gap junction Cx43 on DIND after SAH.Methods:Establishment of blood injection model: caudal venous blood was extracted from normal SD rats after abdominal anaesthesia about 0.3ml was used, occipital cistern puncture was performed under the posture microscope, clear cerebrospinal fluid (CSF) flowed out about 0.1ml, caudal vein blood was injected into caudal vein blood, and the same steps were performed the next day.Complete the whole modeling process.2Identification of SAH model: it was divided into simple SAH model group and normal group (sham-operation group). On the 7th day, the diameter of basilar artery and nerve function score were measured with ink staining method to evaluate the nerve function defect, and the SAH model was identified.3. Detection of virus silencing efficiency: adenovirus cistern cistern injection was used to set up virus treatment group, SAH group and normal control group.Sprague-Dawley rats were killed at four time points (1 d, 3 d, 7 d, 14 d). Western bloting was taken from basilar artery to detect the efficiency of virus silencing Cx43.4. Neurological function score: SAH model group and normal group, preconditioning group and treatment group were divided into 7 days and 14 days to perform nerve function score (modified garcia JH and beam balance scoring method).5MRI examination: normal group and simple SAH model group, preconditioning group and treatment group were examined with MRII-DWII-PWI at 14 days after 7 days, including cerebral blood flow (CBF), apparent diffusion coefficient (ADCC).Results:1. The diameter of basilar artery was detected by Indian ink staining. The diameter of basilar artery in SAH group was significantly smaller than that in sham operation group 7 days after modeling.2, detection of adenovirus silencing efficiency: adenovirus cistern injection can effectively silence the expression of cerebrovascular Cx43.3, nerve function score: the improved garcia score method: compared with the normal group, there was no significant difference between the control group and the control group in 14 days after the onset of neurologic deficit symptoms, all the experimental treatment groups were compared with the simple SAH group, and all the experimental treatment groups were compared with the simple SAH group.There was no significant difference between all groups in 7 days and 14 days.(4) the results of MRI showed that the CBF and ADC decreased on the 7th day compared with the normal group, the virus pretreatment group and virus treatment group could improve the ADC value after 14 days compared with the simple SAH model group, and the glycyroxic acid pretreatment group and the glycyrrhizic acid treatment group could not improve the ADC value after 14 days.Compared with SAH group, virus solvent pretreatment group, virus solvent pretreatment group, glycinic acid solvent pretreatment group and glycinic acid solute pretreatment group could not improve the SAH value at 7 and 14 days.Conclusion:1. The second occipital cistern injection model of autologous caudal vein blood can reproduce the course of SAH.2.Adenoviruses injected into cistern of occipital cistern can improve the CBFs after SAH after down-regulation of Cx43.It can improve the prognosis of SAH.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R651.1

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