自发性动脉压力感受性反射功能缺陷模型大鼠的培育、表型及其缺陷的机制研究

发布时间：2018-05-28 22:17

本文选题：压力感受性反射敏感性 + ABR-DRs　；参考：《第二军医大学》2016年博士论文

【摘要】：动脉压力感受性反射(arterial baroreflex,ABR)可以缓冲血压的升高和降低,是心血管活动最重要的调节机制之一。ABR功能可以被量化,用压力感受性反射敏感性(baroreflex sensitivity,BRS)表示。BRS被认为是自主神经系统的标志。多中心临床试验结果表明,无论有无心血管疾病,BRS都是高血压、脑卒中、心律失常、心力衰竭及心肌梗死等疾病死亡率的强预测因子。当前常用去窦弓神经术破坏ABR传入神经(窦神经和主动脉神经)作为ABR功能缺陷的动物模型。以该动物模型为基础,在ABR功能和心血管疾病的关系及ABR功能低下导致高血压终末器官损伤等发面取得了一定的研究进展。但是该模型的缺点是非自发性,不能真正地模拟ABR功能缺陷的原因,与临床实际相差较大,并不是理想的动物模型。ABR缺陷的具体原因及分子机制现在还不明确,这也导致反射功能低下的基础研究进展缓慢。缺乏自发性ABR缺陷的动物模型是这一问题的限制因素。我们推测培育成功的自发性ABR缺陷动物,也会在许多心血管疾病方面表现出高风险性。基于这一模型,可以进一步明确ABR缺陷的中枢分子靶标。我们利用55只雄性和61只雌性SD大鼠筛选ABR功能正常和低下的大鼠。清醒自由活动状态下BRS和血压的测量结果显示,雄性大鼠的平均BRS和收缩压(systolic blood pressure,SBP)分别为0.77 ms/mm Hg和130 mm Hg。雌雄之间没有差异。根据BRS的分布,我们将BRS0.6 ms/mm Hg的大鼠定义为动脉压力感受性反射功能缺陷的大鼠(arterial baroreflex-deficient rats,ABR-DRs),将BRS0.8ms/mm Hg的大鼠定义为动脉压力感受性反射功能正常的大鼠(arterial baroreflex-normal rats,ABR-NRs)。由于高血压会影响BRS和交感活化,在培育过程中我们只选择血压正常的大鼠进行培育和繁殖。根据上述标准,我们选择了20只(♂:♀=1:1)BRS低的大鼠作为ABR-DRs的原代,20只(♂:♀=1:1)BRS高的大鼠作为ABR-NRs的原代。每一株大鼠原代,均采取随机配对的方法进行交配,得到第一代ABR-DRs,及其对照ABR-NRs。随后代数的大鼠采取“先近亲繁殖后筛选”的方式进行培育,即先兄妹近亲繁殖,待幼鼠离乳后,再对其父本进行清醒动物血压和BRS的测量。只有两个指标均符合上述筛选的标准,其子代才予以保留;否则,子代被淘汰。我们只筛选鉴定雄性动物,以减轻工作负担。经过20代的选择性培育,两株动物BRS差异显著(ABR-NRs vs ABR-DRs,1.20±0.26 ms/mm Hg vs 0.46±0.12 ms/mm Hg,P0.01)。在培育过程中,尽管所有高血压大鼠的子代均已被淘汰,ABR-DRs的血压依然高于ABR-NRs。从第9代起,ABR-DRs的SBP和DBP比ABR-NRs均高约10 mm Hg。除了个别代数,两株大鼠的心率(heart rate,HR)并不存在差异。同时,我们对第19、20代的雌性大鼠也进行了血压和BRS的测量,发现雌雄间各个指标趋势相同。因此,在随后的研究中,我们只选用雄性大鼠。我们在不同年龄段(1、2、4、6、8月龄)测量了两株模型动物的BRS,以检查年龄是否影响两株大鼠间的差异。结果发现,从1月龄起,ABR-DRs的BRS就显著低于ABR-NRs(0.25±0.08 ms/mm Hg vs 0.72±0.25 ms/mm Hg,n=7,P0.01)。随着月龄的增加,两株大鼠的BRS总体均呈增加趋势。到8月龄时,ABR-NRs的BRS高达1.19 ms/mm Hg,ABR-DRs则只增加到0.45 ms/mm Hg(P0.01)。在接下来的研究中,我们开始验证我们的工作假设:心血管疾病的风险性随着BRS的分离而明显不同。我们通过股动、静脉插管的方法动态监测了不同月龄(1、2、4、6、8月龄)两株大鼠的SBP、DBP和心率。结果发现,在1月龄时,ABR-DRs的SBP和DBP就都显著高于ABR-NRs约10 mm Hg,心率显著快于ABR-NRs;随着月龄的增加,两株大鼠的血压持续性缓慢增加,差异依然存在,但心率不再有差异。我们也用尾动脉测压的方法动态监测了不同月龄时的上述指标,得到了相似的结果。更为重要的是,ABR-DRs的高血压发生率显著高于ABR-NRs。在第10代时,ABR-NRs的高血压发生率为10.7%(n=28),而ABR-DRs则为38.9%(n=36,P0.05)。在第19代,差异更为显著(ABR-NRs,0%,n=28;ABR-DRs,57.8%,n=38;P0.01)。这些结果表明ABR-DRs的高血压风险性增加。在2月龄时,我们检测了两株模型大鼠血清中血糖、血脂、肝肾功能相关代谢指标,发现ABR-DRs的随机血糖、空腹血糖、胆固醇、甘油三酯、低密度脂蛋白、高密度脂蛋白和瘦素均显著高于ABR-NRs,表明ABR-DRs出现了血糖、血脂代谢异常。而空腹胰岛素及肝肾功能相关指标没有差异。我们还检测了8月龄大鼠的糖代谢相关指标。与ABR-NRs相比,ABR-DRs的随机血糖、空腹血糖和糖化血红蛋白显著升高。但是,二者的空腹胰岛素水平并没有显著的差异。同时,ABR-DRs的糖耐量和胰岛素耐量受损,但胰岛素的分泌没有问题,很可能是机体对胰岛素产生了抵抗。因此,ABR-DRs存在代谢异常。我们观察记录了第20代ABR-NRs和ABR-DRs在1-25月龄期间的体重和进食量。在1月龄和2月龄时,ABR-DRs的进食量显著高于ABR-NRs,但二者体重没有差异。随后在3-8月龄(6月龄除外)期间,两株大鼠的进食量相同;从9月龄开始,ABR-DRs的进食量显著低于ABR-NRs。从7月龄开始,ABR-DRs的体重持续显著高于ABR-NRs。这两个指标的差异一直持续到24月龄。在10月龄时,我们对两株大鼠进行了脂肪分布的测量。与ABR-NRs相比,ABR-DRs的体重增加了96 g,总脂肪重量增加约45 g,其皮下、附睾、肠系膜、肾周及腹膜后、肩胛骨、胃周和主动脉周围的脂肪重量和比例均显著增加。这些结果表明,ABR-DRs存在超重问题。与ABR-NRs相比,ABR-DRs在舒张期和收缩期的左心室内径、左心室体积及射血体积、射血分数、心输出量均没有显著性差异,表明其并不存在心功能受损;但它们在舒张期和收缩期的左心室前壁厚度、收缩期的左心室后壁厚度显著增加,这表明ABR-DRs存在心肌肥厚。通过称重,我们发现,与ABR-NRs相比,ABR-DRs的心脏、左心室和右心室的重量显著增加,进一步说明其存在心肌肥厚;ABR-DRs单位厘米的主动脉重量也显著增加;二者右肾的重量并没有统计学差异。2月龄大鼠的急性心肌缺血结果显示,ABR-DRs左心室缺血组织重量和面积增加,说明其缺血损伤加重。在阻断大脑中动脉造成缺血性脑卒中模型中,ABR-DRs的缺血面积和比例显著增加,神经缺陷评分也明显增加。这些结果表明ABR-DRs急性心肌梗死和脑缺血的损伤加重。1-16月龄时的负重强迫游泳结果显示,ABR-DRs在各个时间点的游泳时间均短于ABR-NRs;并且随着年龄的增加,两株大鼠的游泳时间逐渐降低。跑台实验的结果也显示ABR-DRs跑步至力竭的时间显著短于ABR-NRs。这些结果说明,ABR-DRs的有氧运动能力低下。我们也记录了ABR-NRs和ABR-DRs的生存时间,结果发现,ABR-DRs的寿命显著缩短。以该动物模型为基础,我们展开了ABR功能缺陷的机制研究。我们通过记录肾交感神经活动(renal sympathetic nerve activity,RSNA)来检测ABR-DRs的交感神经是否存在过度活化。基础状态下,ABR-DRs的RSNA与ABR-NRs并没有统计学差异。然后,我们通过静脉给予一定量的去氧肾上腺素来升高血压(约50 mm Hg),激活压力反射,以观察ABR对交感神经活化的调控。结果表明,ABR-DRs的RSNA变化率与ABR-NRs相比并没有统计学差异。这些结果表明ABR-DRs压力反射的交感支并没有异常。与ABR-NRs相比,升压后ABR-DRs的心率降低幅度显著减小。而心率主要是由迷走神经系统调控的,因此上述结果表明ABR-DRs的副交感功能受损。迷走神经的中枢是疑核。疑核节前心迷走神经元(cardiac vagal preganglionic neurons,CVPNs)决定了心脏的紧张性和反射性调控,其自发性电活动的强弱与疑核副交感功能密切相关。我们利用逆向荧光染料(XRITC,Invitrogen)标记CVPNs,然后进行电生理膜片钳实验。与ABR-NRs相比,ABR-DRs疑核CVPNs的自发性放电频率频率显著降低。加入外源性谷氨酸,可兴奋CVPNs,增加自发性放电的频率,但ABR-DRs增加的百分比显著降低。这些数据表明,ABR-DRs的疑核CVPNs的电活动异常。谷氨酸能和γ-氨基丁酸能突触传递通路是疑核CVPNs的主要突触传入。我们同时阻断NMDA(N-methyl-D-aspartic acid,N-甲基-D-天冬氨酸)受体、AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid,α-氨基-3-羟基-5-甲基-4-异恶唑丙酸)受体和GABAA(γ-aminobutyric acid,γ-氨基丁酸)受体,ABR-NRs和ABR-DRs CVPNs自发性放电的差异被取消。但是单独阻断GABAA受体并不影响两组大鼠CVPNs自发性放电之间的差异。因此,ABR-DRs疑核CVPNs的自发性放电频率降低主要是由谷氨酸能突触通路影响的。AMPA受体阻断剂预处理可以取消两株模型大鼠CVPNs自发性放电频率的差异,并且完全取消了谷氨酸对CVPNs的兴奋作用。NMDA阻断剂并不能取消两株大鼠间的放电差异,也不影响谷氨酸对CVPNs的兴奋作用,并且ABR-DRs放电频率增加的百分比依然显著低于ABR-NRs。因此,AMPA受体参与了ABR-DRs疑核CVPNs的电活动异常。本课题的实验结果表明,心血管疾病的危险因素确实随着BRS的分离而明显不同。ABR-DRs在许多心血管疾病方面表现出高风险性,如高血压、高血糖、高血脂、超重及有氧运动能力低下。BRS受损导致迷走神经传出减弱,是心血管疾病的危险因素,也会显著缩短生存时间。疑核心迷走神经元自发性放电频率降低,对谷氨酸反应减弱,AMPA受体很可能参与了ABR功能受损。
[Abstract]:Arterial baroreflex (ABR) can cushion the increase and decrease of blood pressure. It is one of the most important regulatory mechanisms of cardiovascular activity..ABR function can be quantified. With the baroreflex sensitivity (BRS) sensitivity (baroreflex sensitivity, BRS),.BRS is considered as a sign of the autonomic nervous system. A multicenter clinical trial junction The results show that BRS is a strong predictor of the mortality of hypertension, stroke, arrhythmia, heart failure, and myocardial infarction, regardless of the cardiovascular disease. The current use of DDN to destroy the ABR afferent nerve (sinus nerve and aortic nerve) as an animal model of ABR dysfunction. Based on this animal model, ABR work The relationship with cardiovascular disease and the hypofunction of ABR lead to the development of the end organ damage of hypertension. However, the defect of the model is non spontaneous and can not truly simulate the causes of ABR functional defects, which are different from the clinical practice, and are not the specific causes and molecules of the.ABR defect in the animal model. The mechanism is not yet clear, which has also led to a slow progress in basic research on reflex hypofunction. The animal model lacking spontaneous ABR defects is a limiting factor in this problem. We speculate that a successful spontaneous ABR deficient animal may also be highly risky in many cardiovascular diseases. Based on this model, it can be further developed. The central molecular target of ABR defect was identified. We used 55 male and 61 female SD rats to screen the normal and low function rats of ABR. The results of BRS and blood pressure in conscious free activity showed that the average BRS and systolic blood pressure (systolic blood pressure, SBP) in male rats were 0.77 ms/mm Hg and 130 mm. According to the distribution of BRS, we defined the rats of BRS0.6 ms/mm Hg as the rat (arterial baroreflex-deficient rats, ABR-DRs) of the arterial baroreceptor reflex (arterial baroreflex-deficient rats, ABR-DRs), and defined the rats of BRS0.8ms/mm Hg as a rat with normal arterial baroreceptor reflex (arterial baroreflex-normal). Blood pressure affects BRS and sympathetic activation. In the process of breeding, we only choose rats with normal blood pressure to breed and reproduce. According to the above criteria, we chose 20 rats with low BRS as the original ABR-DRs, and 20 rats with high BRS as the original generation of ABR-NRs. In the method of mating, the first generation ABR-DRs was obtained, and the rats of the ABR-NRs. subsequent algebra were bred by "first close breeding after screening". That is, the siblings were inbred, and then the young rats were separated from the milk, and then the blood pressure and BRS of the waking animals were measured. Only two indexes were in line with the criteria of the above screening. Instead, the progeny was eliminated. We only screened the male animals to reduce the burden of work. After 20 generations of selective breeding, two animals had significant differences in BRS (ABR-NRs vs ABR-DRs, 1.20 + 0.26 ms/mm Hg vs 0.46 + 0.12 ms/mm Hg, P0.01). In the breeding process, all the offspring of the hypertensive rats were eliminated, ABR The blood pressure of -DRs was still higher than that of ABR-NRs. from the ninth generation. The SBP and DBP of ABR-DRs were 10 mm Hg. higher than that of ABR-NRs. The heart rate (heart rate, HR) of two rats did not differ. In the later study, we only chose male rats. We measured the BRS of two model animals at different age groups (1,2,4,6,8 months of age) to check whether the age affected the difference between two rats. The results showed that from 1 month old, the BRS of ABR-DRs was significantly lower than ABR-NRs (0.25 + 0.08 ms/mm Hg vs 0.72 + 0.25 ms/mm Hg, n=7, P0.01). As the age increased, the BRS of the two rats tended to increase. At 8 month old, the BRS of ABR-NRs was 1.19 ms/mm Hg, and ABR-DRs only increased to 0.45 ms/mm Hg (P0.01). In the next study, we began to verify our work hypothesis that the risk of cardiovascular disease was significantly different with the separation of BRS. The method of intubation dynamically monitored the SBP, DBP and heart rate of two rats of different months of age (1,2,4,6,8 months of age). The results showed that the SBP and DBP of ABR-DRs were significantly higher than that of ABR-NRs about 10 mm Hg at 1 month old, and the heart rate was significantly faster than ABR-NRs. With the increase of month age, the blood pressure of two rats increased slowly, but the difference still existed, but heart rate was no longer There was a difference. We also used the tail artery pressure measurement to dynamically monitor the above indicators at different months of age and get similar results. More importantly, the incidence of hypertension in ABR-DRs was significantly higher than that of ABR-NRs. at the tenth generation. The incidence of hypertension in ABR-NRs was 10.7% (n=28), while ABR-DRs was 38.9% (n=36, P0.05). In the nineteenth generation, the difference was more significant. The results were significant (ABR-NRs, 0%, n=28; ABR-DRs, 57.8%, n=38; P0.01). These results showed an increase in the risk of hypertension in ABR-DRs. At 2 month old, we detected blood glucose, blood lipid, liver and kidney function related metabolic indicators in two model rats, and found the random blood sugar, fasting blood glucose, cholesterol, triglyceride, LDL, high density of ABR-DRs. Both lipoprotein and leptin were significantly higher than ABR-NRs, indicating that ABR-DRs had blood glucose and abnormal metabolism of blood lipids. There was no difference in fasting insulin and liver and kidney function related indexes. We also detected the glucose metabolism related indexes in 8 month old rats. Compared with ABR-NRs, the random blood sugar, empty abdomen blood glucose and glycated hemoglobin were significantly higher than that of ABR-NRs. But, two There was no significant difference in the level of fasting insulin. At the same time, ABR-DRs's glucose tolerance and insulin tolerance were impaired, but insulin secretion was not a problem. It was probably the body's resistance to insulin. Therefore, there was a metabolic abnormality in ABR-DRs. We observed the weight and eating of the twentieth generation of ABR-NRs and ABR-DRs during the period of 1-25 months of age. At 1 month old and 2 month old, the intake of ABR-DRs was significantly higher than that of ABR-NRs, but there was no difference in weight between the two and the 3-8 months of age (except for 6 month old), and the intake of the two rats was the same; from 9 month old, the intake of ABR-DRs was significantly lower than that of ABR-NRs. from 7 month old, and the weight of ABR-DRs was significantly higher than that of ABR-NRs., the two indicators. The difference continued to 24 month old. At 10 month old, we measured the fat distribution in two rats. Compared with ABR-NRs, the weight of ABR-DRs increased by 96 g and the total fat weight increased by about 45 g. The weight and proportion of fat around the stomach and around the stomach and aorta increased significantly in the subcutaneous, epididymis, mesentery, perirenal and retroperitoneal, scapula, and the aorta. The results showed that ABR-DRs was overweight. Compared with ABR-NRs, ABR-DRs had no significant difference in the diastolic and systolic left ventricular diameter, left ventricular volume, ejection volume, ejection fraction, and cardiac output, indicating that there was no impairment of cardiac function, but they were in systolic and systolic left ventricular anterior wall thickness and left systolic left The thickness of the posterior wall of the ventricle increased significantly, which indicates that ABR-DRs has cardiac hypertrophy. By weighing, we found that the weight of the heart, the left ventricle and the right ventricle of the ABR-DRs increased significantly compared with ABR-NRs, further indicating the existence of cardiac hypertrophy; the weight of the aorta in ABR-DRs centimeters was also significantly increased; and the weight of the right kidney was not statistically significant in two. The results of acute myocardial ischemia in.2 month old rats showed an increase in the weight and area of ABR-DRs left ventricular ischemic tissue, indicating that the ischemic injury was aggravated. The ischemic area and proportion of ABR-DRs increased significantly in the occlusion of the middle cerebral artery, and the neurological deficit score increased significantly. These results showed that the ABR-DRs was acute. The results showed that the swimming time of ABR-DRs at all time points was shorter than that of ABR-NRs when the injury of myocardial infarction and cerebral ischemia aggravated the.1-16 months of age, and the swimming time of two rats decreased gradually with the increase of age. The result of the runway experiment also showed that the time of ABR-DRs running to exhaustion was significantly shorter than that of ABR-NRs.. The results showed that the aerobic activity of ABR-DRs was low. We also recorded the survival time of ABR-NRs and ABR-DRs, and found that the life span of ABR-DRs was significantly shortened. Based on the animal model, we developed the mechanism of the ABR function defect. We checked the renal sympathetic activity (renal sympathetic nerve activity, RSNA). There was no significant activation in the sympathetic nerve of ABR-DRs. In the base state, there was no statistical difference between the RSNA of ABR-DRs and ABR-NRs. Then, we administered a certain amount of deoxyadrenaline to elevate hypertension (about 50 mm Hg) to activate the pressure reflex to observe the regulation of the sympathetic activation of ABR. The results showed that the RSNA change of ABR-DRs. There was no statistical difference compared with ABR-NRs. These results showed that the sympathetic branches of the ABR-DRs pressure reflex were not abnormal. Compared with ABR-NRs, the heart rate decreased significantly after the boost, and the heart rate was mainly regulated by the vagus system. Therefore, the above results indicate that the parasympathetic function of ABR-DRs is impaired. The vagus nerve is impaired. The center is the nucleus. Cardiac vagal preganglionic neurons (CVPNs) determines the tension and reflex regulation of the heart. The intensity of spontaneous electrical activity is closely related to the parasympathetic parasympathetic function. We use the reverse fluorescent dye (XRITC, Invitrogen) to mark CVPNs and then carry out the electrophysiological patch clamp experiment. Compared with ABR-NRs, the spontaneous discharge frequency of ABR-DRs CVPNs was significantly reduced. Adding exogenous glutamic acid could excite CVPNs and increase the frequency of spontaneous discharge, but the percentage of ABR-DRs increased significantly. These data indicate that the electrical activity of ABR-DRs in the nuclear CVPNs is abnormal. Glutamate and gamma aminobutyric acid can transfer through the synapse. The pathway is the main synapse afferent of the nuclear CVPNs. We also block the NMDA (N-methyl-D-aspartic acid, N- methyl -D- aspartic acid) receptor, AMPA (alpha -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, alpha amino -3- hydroxyl -5- methyl -4- isooxazole propionic acid) receptor and the receptor of gamma aminobutyric acid (gamma aminobutyric acid) The difference between the spontaneous discharge of s CVPNs was cancelled. But the isolation of GABAA receptor alone did not affect the difference between the spontaneous discharge of the two groups of rats. Therefore, the spontaneous discharge frequency of CVPNs in the ABR-DRs nuclear CVPNs was mainly caused by the preconditioning of the.AMPA receptor blocker, which was influenced by the glutamic acid synaptic pathway, and the CVPNs self of the two model rats could be cancelled. The difference in the frequency of hair discharge, and completely abolished the excitatory effect of glutamic acid on CVPNs,.NMDA blocker did not cancel the discharge difference between two rats, and did not affect the excitatory effect of glutamic acid on CVPNs, and the percentage of the ABR-DRs discharge frequency increased significantly lower than that of ABR-NRs., so the AMPA receptor was involved in ABR-DRs suspected CVPNs. The results of this study showed that the risk factors of cardiovascular disease did differ significantly with the separation of BRS and.ABR-DRs showed high risk in many cardiovascular diseases, such as hypertension, hyperglycemia, hyperlipidemia, overweight and impaired aerobic activity.BRS, which resulted in the weakening of the vagus nerve, which was the cardiovascular disease. The risk factors of the disease also significantly shorten the survival time. The spontaneous discharge frequency of the nucleus vagus neurons is reduced, the response to glutamic acid is weakened, and the AMPA receptor is likely to be involved in the impairment of ABR function.
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R-332;R54

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