本文選題：主動(dòng)脈夾層 + 基質(zhì)金屬蛋白酶-9�。� 參考：《福建醫(yī)科大學(xué)》2016年博士論文

【摘要】：主動(dòng)脈夾層(aortic dissection,AD)是指主動(dòng)脈內(nèi)膜局部形成破口,高速血流沖擊使內(nèi)膜/中膜剝離擴(kuò)展造成主動(dòng)脈壁中層沿長(zhǎng)軸分離,形成真假兩腔的危重主動(dòng)脈疾病。該病的特點(diǎn)是進(jìn)展快,病情兇險(xiǎn),缺乏有效的藥物根治,外科手術(shù)治療是唯一根治手段。高血壓是AD最常見的基礎(chǔ)疾病,降血壓是預(yù)防和治療AD的重要手段。血壓升高導(dǎo)致血管壁機(jī)械張力升高,最終導(dǎo)致AD的發(fā)生,但是血壓升高的程度和高血壓的發(fā)病時(shí)間對(duì)AD形成的影響,以及高血壓誘導(dǎo)AD發(fā)生的具體機(jī)理并不清楚。近年來(lái),隨著人們對(duì)主動(dòng)脈夾層發(fā)病機(jī)制的深入研究,發(fā)現(xiàn)基質(zhì)金屬蛋白酶-9(matrix metalloproteinase-9,MMP-9)與AD的發(fā)病關(guān)系密切,其含量及活性的增加,可過(guò)度降解主動(dòng)脈壁細(xì)胞外基質(zhì),導(dǎo)致主動(dòng)脈中層退行性變。研究證實(shí)機(jī)械張力能誘導(dǎo)平滑肌細(xì)胞表達(dá)MMP-9。但是血壓的升高,機(jī)體如何將這種機(jī)械信號(hào)轉(zhuǎn)化為生物信號(hào),這個(gè)過(guò)程不明確。為了研究這一機(jī)制,我們建立了主動(dòng)脈夾層模型,探討機(jī)械牽張力度和牽拉時(shí)間與MMP-9表達(dá)量之間的關(guān)系,以及機(jī)械張力誘導(dǎo)主動(dòng)脈夾層血管中MMP-9表達(dá)的分子機(jī)制。第一部分:研究不同機(jī)械張力和牽拉時(shí)間對(duì)主動(dòng)脈夾層血管MMP-9表達(dá)水平的影響目的:研究在離體情況下,使用不同的機(jī)械張力和不同的牽拉時(shí)間對(duì)大鼠腹主動(dòng)脈夾層血管MMP-9表達(dá)水平的影響以及不同的機(jī)械張力對(duì)人主動(dòng)脈夾層血管MMP-9表達(dá)水平的影響。方法:以成年雄性Sprague Dawley(SD)大鼠為研究對(duì)象(體重250g±30g,n=30,分為6組,每組5只),采用動(dòng)脈壁間注射豬胰彈力蛋白酶法來(lái)制備大鼠腹主動(dòng)脈夾層動(dòng)物模型,并運(yùn)用多普勒超聲來(lái)證實(shí)。建立大鼠腹主動(dòng)脈夾層模型48h后,取出腹主動(dòng)脈夾層血管,制備血管環(huán),去除動(dòng)脈環(huán)的內(nèi)膜和外膜,在離體組織恒溫灌流室中,第1~4組分別用0g、1g、3g、5g的機(jī)械張力牽拉血管環(huán),牽拉30min后,收集標(biāo)本;第5~6組均用3g張力牽拉血管環(huán),牽拉時(shí)間分別為1h、2h,收集標(biāo)本;用免疫印跡法(Western-Blot)檢測(cè)MMP-9的表達(dá)量。將10例Stanford A型AD患者的主動(dòng)脈夾層血管片制作成小段血管環(huán),分別用0g、3g、4g、5g、6g、7g、8g、9g張力牽拉血管環(huán)30min,收集標(biāo)本,用Western blot檢測(cè)標(biāo)本中的MMP-9表達(dá)水平。結(jié)果:在動(dòng)物模型制作24小時(shí)后,運(yùn)用多普勒超聲可證實(shí)大鼠腹主動(dòng)脈夾層形成。通過(guò)Western blot法檢測(cè)表明,大鼠腹主動(dòng)脈夾層血管在1g,3g,5g張力作用下,MMP-9的表達(dá)量增加,表達(dá)水平隨張力的變大而上調(diào),在3g時(shí)表達(dá)量達(dá)到高峰,而在5g時(shí)表達(dá)量有所回落,且與牽拉時(shí)間并不呈正相關(guān)。在人主動(dòng)脈夾層血管中,Western blot結(jié)果顯示:4~6g牽張力能使人主動(dòng)脈夾層血管的MMP-9表達(dá)水平進(jìn)一步上調(diào),而大于8g的機(jī)械張力卻并不能增加MMP-9表達(dá)量。結(jié)論:1.動(dòng)脈壁間注射豬胰彈力蛋白酶是制作大鼠腹主動(dòng)脈夾層動(dòng)物模型的良好方法。2.在離體情況下,機(jī)械張力可以誘導(dǎo)大鼠腹主動(dòng)脈血管表達(dá)MMP-9,而且在3g的機(jī)械張力作用下,表達(dá)量可達(dá)到高峰,且與牽拉時(shí)間不成正相關(guān)。3.人主動(dòng)脈夾層血管在4~6g機(jī)械張力作用下可使MMP-9表達(dá)水平上調(diào),而大于8g的機(jī)械張力卻并不增加其MMP-9表達(dá)量。第二部分:探討機(jī)械張力通過(guò)牽張離子通道誘導(dǎo)大鼠腹主動(dòng)脈夾層血管MMP-9表達(dá)的分子機(jī)制目的:探討機(jī)械牽張誘導(dǎo)大鼠腹主動(dòng)脈夾層血管表達(dá)MMP-9的分子機(jī)制,從而為研究AD的發(fā)病機(jī)制提供新的視野。方法:采用動(dòng)脈壁間注射豬胰彈力蛋白酶法來(lái)制備大鼠腹主動(dòng)脈夾層動(dòng)物模型,并運(yùn)用多普勒超聲來(lái)證實(shí)。80只成年雄性SD大鼠(體重250g±30g)隨機(jī)平均分成兩大組:腹主動(dòng)脈夾層組(n=40)、假手術(shù)組(n=40),分別在術(shù)前、術(shù)后第1、3、5、7、14、21、30天抽取大鼠血液,用酶聯(lián)免疫吸附法(ELISA法)檢測(cè)其血漿MMP-9表達(dá)量。成年雄性SD大鼠(體重250g±30g,n=40,分為8組,每組5只),建立腹主動(dòng)脈夾層動(dòng)物模型48h后,取出腹主動(dòng)脈夾層血管,制備血管環(huán),去除動(dòng)脈環(huán)的內(nèi)膜和外膜,分成8個(gè)組(0g組、1g組、3g組、5g組、3g+三氯化釓組、3g+鏈霉素組、3g+SN50組、3g+SN50M組),并分別用0g、1g、3g、5g、3g、3g、3g、3g張力牽拉各組血管環(huán)30min,收集標(biāo)本,分別用Western blot法和熒光定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)法(real time RT-PCR)檢測(cè)8組標(biāo)本中的MMP-9蛋白/m RNA表達(dá)情況,以及用ELISA法檢測(cè)使用三氯化釓、鏈霉素、SN50、SN50M預(yù)處理過(guò)的4組血管環(huán)的核因子κappa B(NF-κB)P65的表達(dá)情況。結(jié)果:在大鼠腹主動(dòng)脈夾層形成后,MMP-9在血漿水平的表達(dá)量隨時(shí)間的延長(zhǎng)而增加,并在術(shù)后1周達(dá)到頂峰,且長(zhǎng)時(shí)間地維持較高表達(dá)量。在大鼠腹主動(dòng)脈夾層血管中,Western blot和熒光定量PCR顯示:1g組,3g組,5g組中的MMP-9大量表達(dá),且3g組為表達(dá)高峰;用牽張離子通道(stretchactivated ion channel,SAC)阻斷劑(三氯化釓、鏈霉素)預(yù)處理過(guò)的動(dòng)脈環(huán)中MMP-9表達(dá)量大大降低,而NF-κB阻滯劑(SN50)由于阻斷了NF-κB的活化,亦可抑制夾層血管表達(dá)MMP-9,但SN50的無(wú)活性對(duì)照肽(SN50M)則無(wú)這種作用;通過(guò)ELISA法表明:機(jī)械張力可增加大鼠腹主動(dòng)脈夾層血管NF-κB的活性,而SAC阻斷劑(三氯化釓、鏈霉素)和NF-κB阻滯劑(SN50)可阻斷NF-κB的活性,而SN50M則不能抑制其活性。結(jié)論:1.大鼠腹主動(dòng)脈夾層形成后,血漿中的MMP-9表達(dá)量隨時(shí)間延長(zhǎng)而增加。2.大鼠腹主動(dòng)脈夾層血管在機(jī)械張力誘導(dǎo)下,可大量表達(dá)MMP-9,這種作用機(jī)制可能是機(jī)械張力作用于夾層血管后,通過(guò)開放血管平滑肌細(xì)胞上的SAC,進(jìn)而活化NF-κB,從而在轉(zhuǎn)錄水平調(diào)節(jié)MMP-9的表達(dá)。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R543.1

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