机械牵张对主动脉夹层血管MMP-9表达的影响及其分子机制研究

发布时间：2018-05-26 18:22

本文选题：主动脉夹层 + 基质金属蛋白酶-9　；参考：《福建医科大学》2016年博士论文

【摘要】：主动脉夹层(aortic dissection,AD)是指主动脉内膜局部形成破口,高速血流冲击使内膜/中膜剥离扩展造成主动脉壁中层沿长轴分离,形成真假两腔的危重主动脉疾病。该病的特点是进展快,病情凶险,缺乏有效的药物根治,外科手术治疗是唯一根治手段。高血压是AD最常见的基础疾病,降血压是预防和治疗AD的重要手段。血压升高导致血管壁机械张力升高,最终导致AD的发生,但是血压升高的程度和高血压的发病时间对AD形成的影响,以及高血压诱导AD发生的具体机理并不清楚。近年来,随着人们对主动脉夹层发病机制的深入研究,发现基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)与AD的发病关系密切,其含量及活性的增加,可过度降解主动脉壁细胞外基质,导致主动脉中层退行性变。研究证实机械张力能诱导平滑肌细胞表达MMP-9。但是血压的升高,机体如何将这种机械信号转化为生物信号,这个过程不明确。为了研究这一机制,我们建立了主动脉夹层模型,探讨机械牵张力度和牵拉时间与MMP-9表达量之间的关系,以及机械张力诱导主动脉夹层血管中MMP-9表达的分子机制。第一部分:研究不同机械张力和牵拉时间对主动脉夹层血管MMP-9表达水平的影响目的:研究在离体情况下,使用不同的机械张力和不同的牵拉时间对大鼠腹主动脉夹层血管MMP-9表达水平的影响以及不同的机械张力对人主动脉夹层血管MMP-9表达水平的影响。方法:以成年雄性Sprague Dawley(SD)大鼠为研究对象(体重250g±30g,n=30,分为6组,每组5只),采用动脉壁间注射猪胰弹力蛋白酶法来制备大鼠腹主动脉夹层动物模型,并运用多普勒超声来证实。建立大鼠腹主动脉夹层模型48h后,取出腹主动脉夹层血管,制备血管环,去除动脉环的内膜和外膜,在离体组织恒温灌流室中,第1~4组分别用0g、1g、3g、5g的机械张力牵拉血管环,牵拉30min后,收集标本;第5~6组均用3g张力牵拉血管环,牵拉时间分别为1h、2h,收集标本;用免疫印迹法(Western-Blot)检测MMP-9的表达量。将10例Stanford A型AD患者的主动脉夹层血管片制作成小段血管环,分别用0g、3g、4g、5g、6g、7g、8g、9g张力牵拉血管环30min,收集标本,用Western blot检测标本中的MMP-9表达水平。结果:在动物模型制作24小时后,运用多普勒超声可证实大鼠腹主动脉夹层形成。通过Western blot法检测表明,大鼠腹主动脉夹层血管在1g,3g,5g张力作用下,MMP-9的表达量增加,表达水平随张力的变大而上调,在3g时表达量达到高峰,而在5g时表达量有所回落,且与牵拉时间并不呈正相关。在人主动脉夹层血管中,Western blot结果显示:4~6g牵张力能使人主动脉夹层血管的MMP-9表达水平进一步上调,而大于8g的机械张力却并不能增加MMP-9表达量。结论:1.动脉壁间注射猪胰弹力蛋白酶是制作大鼠腹主动脉夹层动物模型的良好方法。2.在离体情况下,机械张力可以诱导大鼠腹主动脉血管表达MMP-9,而且在3g的机械张力作用下,表达量可达到高峰,且与牵拉时间不成正相关。3.人主动脉夹层血管在4~6g机械张力作用下可使MMP-9表达水平上调,而大于8g的机械张力却并不增加其MMP-9表达量。第二部分:探讨机械张力通过牵张离子通道诱导大鼠腹主动脉夹层血管MMP-9表达的分子机制目的:探讨机械牵张诱导大鼠腹主动脉夹层血管表达MMP-9的分子机制,从而为研究AD的发病机制提供新的视野。方法:采用动脉壁间注射猪胰弹力蛋白酶法来制备大鼠腹主动脉夹层动物模型,并运用多普勒超声来证实。80只成年雄性SD大鼠(体重250g±30g)随机平均分成两大组:腹主动脉夹层组(n=40)、假手术组(n=40),分别在术前、术后第1、3、5、7、14、21、30天抽取大鼠血液,用酶联免疫吸附法(ELISA法)检测其血浆MMP-9表达量。成年雄性SD大鼠(体重250g±30g,n=40,分为8组,每组5只),建立腹主动脉夹层动物模型48h后,取出腹主动脉夹层血管,制备血管环,去除动脉环的内膜和外膜,分成8个组(0g组、1g组、3g组、5g组、3g+三氯化钆组、3g+链霉素组、3g+SN50组、3g+SN50M组),并分别用0g、1g、3g、5g、3g、3g、3g、3g张力牵拉各组血管环30min,收集标本,分别用Western blot法和荧光定量逆转录聚合酶链反应法(real time RT-PCR)检测8组标本中的MMP-9蛋白/m RNA表达情况,以及用ELISA法检测使用三氯化钆、链霉素、SN50、SN50M预处理过的4组血管环的核因子κappa B(NF-κB)P65的表达情况。结果:在大鼠腹主动脉夹层形成后,MMP-9在血浆水平的表达量随时间的延长而增加,并在术后1周达到顶峰,且长时间地维持较高表达量。在大鼠腹主动脉夹层血管中,Western blot和荧光定量PCR显示:1g组,3g组,5g组中的MMP-9大量表达,且3g组为表达高峰;用牵张离子通道(stretchactivated ion channel,SAC)阻断剂(三氯化钆、链霉素)预处理过的动脉环中MMP-9表达量大大降低,而NF-κB阻滞剂(SN50)由于阻断了NF-κB的活化,亦可抑制夹层血管表达MMP-9,但SN50的无活性对照肽(SN50M)则无这种作用;通过ELISA法表明:机械张力可增加大鼠腹主动脉夹层血管NF-κB的活性,而SAC阻断剂(三氯化钆、链霉素)和NF-κB阻滞剂(SN50)可阻断NF-κB的活性,而SN50M则不能抑制其活性。结论:1.大鼠腹主动脉夹层形成后,血浆中的MMP-9表达量随时间延长而增加。2.大鼠腹主动脉夹层血管在机械张力诱导下,可大量表达MMP-9,这种作用机制可能是机械张力作用于夹层血管后,通过开放血管平滑肌细胞上的SAC,进而活化NF-κB,从而在转录水平调节MMP-9的表达。
[Abstract]:Aortic dissection (AD) refers to the localized rupture of the intima of the aorta. High velocity blood flow impact causes the intimal / middle membrane exfoliation and expansion to separate the middle layer of the aortic wall along the long axis to form a critical aortic disease in the true and false two cavities. The disease is characterized by rapid progress, a dangerous condition, a lack of effective radical cure, and surgical treatment is the only one. A radical cure. Hypertension is the most common basic disease of AD. Lowering blood pressure is an important means to prevent and treat AD. The increase of blood pressure leads to the increase of mechanical tension of the blood vessel wall, which eventually leads to the occurrence of AD, but the extent of the rise of blood pressure and the influence of the time of hypertension on the formation of AD, and the specific mechanism of the occurrence of AD induced by hypertension are not clear. In recent years, with the in-depth study of the pathogenesis of aortic dissection, it is found that the matrix metalloproteinase -9 (matrix metalloproteinase-9, MMP-9) is closely related to the pathogenesis of AD. The increase of its content and activity can degrade the extracellular matrix of the aortic wall and lead to the degeneration of the middle layer of the aorta. In order to study this mechanism, we established a model of aortic dissection to explore the relationship between mechanical stretch and pulling time and the expression of MMP-9, as well as the mechanical tension induced aortic clamp in order to study the mechanism. The molecular mechanism of MMP-9 expression in the interlayer vessel. Part 1: To study the effect of different mechanical tension and stretch time on the level of MMP-9 expression in aortic dissection vessels: To study the effect of different mechanical tension and different pulling time on the MMP-9 expression level of the abdominal aorta in vitro. The effect of mechanical tension on the expression level of MMP-9 in human aortic dissection. Methods: adult male Sprague Dawley (SD) rats were studied (weight 250g + 30g, n=30, divided into 6 groups, 5 rats in each group). The animal model of abdominal aortic dissection was prepared by intramural injection of pig pancreatic elastase, and the Doppler ultrasound was used to confirm the animal model. After the rat abdominal aortic dissection model 48h was established, the abdominal aorta dissection vessel was removed and the vascular ring was prepared. The intima and outer membrane of the artery ring were removed. In the isolated tissue constant temperature perfusion room, group 1~4 pulled the vascular ring with the mechanical tension of 0g, 1g, 3G and 5g respectively. After pulling 30min, the specimens were collected, and the 5~6 group pulled the blood vessel ring with 3G tension and pulled the time. 1H, 2h, samples were collected, and the expression of MMP-9 was detected by immunoblotting (Western-Blot). The aortic dissection of 10 cases of Stanford A AD patients was made into small segment vascular rings. 24 hours after the animal model was made, Doppler ultrasound could be used to confirm the formation of abdominal aortic dissection in rats. By Western blot method, the expression of MMP-9 was increased under the action of 1g, 3G and 5g, the expression level of the rat aorta was increased with the tension, and the expression reached the peak at 3G, while the expression at 5g was expressed. In the aortic dissection, Western blot results showed that 4~6g traction could further increase the level of MMP-9 expression in the aortic dissection, but the mechanical tension greater than 8g did not increase the MMP-9 expression. Conclusion: 1. intra arterial wall injection of pig pancreatic elastase is made. A good method of animal model of abdominal aortic dissection in rats.2. in vitro, mechanical tension can induce the expression of MMP-9 in the aorta of abdominal aorta in rats, and the expression can reach the peak under the action of mechanical tension of 3G, and there is no positive correlation with the pulling time of the.3. human aortic pinch vessel under the action of 4~6g mechanical tension to make MMP-9 expression The mechanical tension that is greater than 8g does not increase the expression of MMP-9. Second part: To explore the molecular mechanism of MMP-9 expression of abdominal aortic dissection induced by mechanical tension through the distraction ion channel in rats: To explore the molecular mechanism of MMP-9 in the abdominal aortic dissection induced by mechanical distraction in rats, so as to study AD The pathogenesis provided a new field of vision. Methods: the rat model of abdominal aortic dissection was prepared by intramural injection of porcine pancreatic elastase, and Doppler ultrasound was used to confirm that.80 adult male SD rats (250g + 30g) were randomly divided into two groups: abdominal active vein dissection group (n=40), sham operation group (n=40), before operation, respectively. The blood of rats was extracted at the 1,3,5,7,14,21,30 day after the operation, and the expression of MMP-9 in plasma was detected by enzyme linked immunosorbent assay (ELISA method). Adult male SD rats (weight 250g + 30g, n=40, divided into 8 groups, 5 rats in each group), after establishing an abdominal aortic dissection animal model 48h, take out the abdominal active vein interlayer vessel, prepare the vascular ring, remove the intima and outer membrane of the artery ring. They were divided into 8 groups (Group 0g, group 1g, group 3G, group 5g, 3g+ three gadolinium chloride group, 3g+ streptomycin group, 3g+SN50 group, 3g+SN50M group), and the collection specimens were collected with 0g, 1g, 3G, 5g, 3G, respectively. The expression of /m RNA and the expression of nuclear factor kappa AppA B (NF- KF) P65 of the 4 groups of vascular rings pretreated with gadolinium chloride, streptomycin, SN50 and SN50M using ELISA method. Results: after the formation of abdominal aortic dissection in rats, MMP-9 in the plasma level increased at any time and reached the peak at 1 weeks after the operation and long after the operation. In the abdominal aortic dissection, Western blot and fluorescence quantitative PCR showed that MMP-9 in group 1g, 3G group, 5g group was expressed in large amount, and 3G was the peak of expression, and the amount of expression in the arterial ring pretreated with the distraction ion channel (stretchactivated ion channel, SAC) blocker (three gadolinium chloride, streptomycin) was large The NF- kappa B blocker (SN50), which blocked the activation of NF- kappa B, also inhibited the expression of MMP-9 in the interlayer blood vessel, but the non active control peptide (SN50M) of SN50 had no such effect. The ELISA method indicated that mechanical tension could increase the activity of NF- kappa B in the abdominal aortic dissection of rats, and the SAC blocker (three gadolinium chloride, streptomycin) and kappa blockage were blocked. The activity of NF- kappa B can be blocked by agent (SN50), while SN50M does not inhibit its activity. Conclusion: after the formation of abdominal aortic dissection in 1. rats, the expression of MMP-9 in plasma increases with time and increases the abdominal aortic dissection vessels in.2. rats under the induction of mechanical tension, and can express MMP-9 in large quantities. This mechanism may be the mechanism of mechanical tension on interlayer blood vessels. After that, the SAC of vascular smooth muscle cells was opened to activate NF- kappa B, thereby regulating the expression of MMP-9 at the transcriptional level.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.1

【参考文献】