本文選題：克魯佩爾因子2 + 血流動(dòng)力學(xué)��； 參考：《第二軍醫(yī)大學(xué)》2014年博士論文

【摘要】：第一部分KLF2在血流動(dòng)力學(xué)誘導(dǎo)的動(dòng)脈瘤動(dòng)物模型中的表達(dá) 目的：觀察不同方法構(gòu)建的血流動(dòng)力學(xué)誘導(dǎo)動(dòng)脈瘤模型中克魯佩爾因子2（KLF2）的表達(dá)及與基底動(dòng)脈血流速度變化的相關(guān)性。 方法：雄性新西蘭白兔144只，隨機(jī)分成三組：假手術(shù)組、單側(cè)頸總動(dòng)脈結(jié)扎組、雙側(cè)頸總動(dòng)脈結(jié)扎組，每組48只，每組各分為術(shù)后1周、2周、3周和4周組，每組12只。假手術(shù)組僅切開(kāi)頸部暴露頸總動(dòng)脈不結(jié)扎，單側(cè)結(jié)扎組統(tǒng)一結(jié)扎右側(cè)頸總動(dòng)脈，雙側(cè)結(jié)扎組進(jìn)行雙側(cè)頸總動(dòng)脈結(jié)扎。各組分別在術(shù)后1周、2周、3周、4周行經(jīng)顱多普勒超聲測(cè)量基底動(dòng)脈血流速度。然后在各時(shí)間點(diǎn)處死動(dòng)物，每組每個(gè)時(shí)間點(diǎn)取6只基底動(dòng)脈分叉部進(jìn)行HE染色、EVG染色、Masson染色、KLF2免疫組化染色等病理檢測(cè)，另外6只基底動(dòng)脈行KLF2western blot蛋白分析。 結(jié)果：各組基底動(dòng)脈平均血流速度測(cè)量結(jié)果，在假手術(shù)組術(shù)后不同時(shí)間點(diǎn)變化不明顯，單側(cè)頸總動(dòng)脈結(jié)扎組術(shù)后血流速度較假手術(shù)組增加，但統(tǒng)計(jì)學(xué)結(jié)果提示無(wú)顯著差異（P＞.05）。雙側(cè)頸總動(dòng)脈結(jié)扎組流速明顯快于假手術(shù)組和單側(cè)頸總動(dòng)脈結(jié)扎組，有顯著統(tǒng)計(jì)學(xué)意義（p＜0.05）。病理學(xué)檢查結(jié)果提示按照動(dòng)脈瘤評(píng)價(jià)標(biāo)準(zhǔn)，假手術(shù)組基底動(dòng)脈尖端管腔平滑，內(nèi)皮細(xì)胞完整，內(nèi)彈力膜完整，無(wú)斷裂和變薄，全組無(wú)動(dòng)脈瘤形成。右側(cè)結(jié)扎組中24只動(dòng)物模型中有1只基底動(dòng)脈尖端略向外膨出，但膨出程度比較低。雙側(cè)結(jié)扎組中有12只向外凸出的動(dòng)脈瘤樣突起，統(tǒng)計(jì)分析表明三組間具有顯著統(tǒng)計(jì)學(xué)差異（p＜0.05）。KLF2western blot定量分析提示假手術(shù)組基底動(dòng)脈的KLF2蛋白表達(dá)隨時(shí)間變化無(wú)明顯變化，而單側(cè)結(jié)扎組蛋白表達(dá)隨時(shí)間變化有所增加，但增加較少。雙側(cè)結(jié)扎組KLF2蛋白表達(dá)在術(shù)后1W起即有顯著增加，隨后一直維持在較高水平，統(tǒng)計(jì)結(jié)果顯示與假手術(shù)組和單側(cè)結(jié)扎組相比具有統(tǒng)計(jì)學(xué)意義(P＜0.05)。KLF2蛋白表達(dá)與血流速度變化相關(guān)分析表明二者具有顯著正相關(guān)性（r=0.845， P＜0.05）。 結(jié)論：1、采用兔雙側(cè)頸總動(dòng)脈結(jié)扎法建立的單純血流動(dòng)力學(xué)誘導(dǎo)的動(dòng)脈瘤模型引起的血流動(dòng)力學(xué)變化最為明顯，更適合做為動(dòng)脈瘤研究的動(dòng)物模型。 2、KLF2的表達(dá)變化與血流速度變化具有一致性，相關(guān)分析提示二者明顯正相關(guān)，，提示其表達(dá)受血流動(dòng)力學(xué)調(diào)控。 第二部分KLF2上游調(diào)控因子miR-92a在血流動(dòng)力學(xué)誘導(dǎo)動(dòng)脈瘤動(dòng)物模型中的表達(dá) 目的：利用不同方法構(gòu)建的血流動(dòng)力學(xué)誘導(dǎo)的動(dòng)脈瘤模型，采用RT-PCR法檢測(cè)各組基底動(dòng)脈分叉部miRNA-92a的表達(dá)差異，探討其對(duì)KLF2的表達(dá)的調(diào)控作用。 方法：雄性新西蘭白兔72只，隨機(jī)分成假手術(shù)組、單側(cè)頸總動(dòng)脈結(jié)扎組、雙側(cè)頸總動(dòng)脈結(jié)扎組各24只，每組各分為術(shù)后1周、2周、3周和4周組，每組6只。然后在各時(shí)間點(diǎn)處死動(dòng)物，取基底動(dòng)脈分叉部進(jìn)行RT-PCR檢測(cè)miRNA-92a的表達(dá)差異。其表達(dá)結(jié)果與基底動(dòng)脈血流速度進(jìn)行相關(guān)分析。 結(jié)果：?jiǎn)蝹?cè)結(jié)扎組和雙側(cè)結(jié)扎組基底動(dòng)脈分叉部mir-92a mRNA表達(dá)水平較假手術(shù)組明顯降低。雙側(cè)結(jié)扎組降低最為明顯，與假手術(shù)組和單側(cè)結(jié)扎組相比具有顯著統(tǒng)計(jì)學(xué)差異(P＜0.05)。mir-92a的mRNA表達(dá)水平與KLF2蛋白表達(dá)相關(guān)分析表明二者顯著呈負(fù)相關(guān)（r=-0.768, P＜0.01），而與基底動(dòng)脈血流速度相關(guān)分析提示二者亦呈負(fù)相關(guān)（r=-0.709, P＜0.01）。 結(jié)論： miRNA-92a mRNA表達(dá)隨血流速度的增加而表達(dá)下降，且與血流速度的變化具有負(fù)相關(guān)性；說(shuō)明miRNA-92a mRNA表達(dá)受血流動(dòng)力學(xué)調(diào)控。miRNA-92a表達(dá)與KLF2表達(dá)趨勢(shì)相反，二者呈顯著負(fù)相關(guān)。 第三部分辛伐他汀對(duì)KLF2及下游因子在動(dòng)脈瘤模型中的表達(dá)影響 目的：探討KLF2/eNOS、VCAM-1、MMP-9通路在動(dòng)脈瘤發(fā)生中的作用機(jī)制及辛伐汀抑制動(dòng)脈瘤生長(zhǎng)的作用。 方法：新西蘭白兔72只，隨機(jī)分成二組：雙側(cè)頸總動(dòng)脈結(jié)扎后辛伐他汀干預(yù)組、雙側(cè)頸總動(dòng)脈結(jié)扎組各36只，每組各分為術(shù)后1周、4周，每組18只。辛伐他汀組自動(dòng)物雙側(cè)頸總動(dòng)脈結(jié)扎術(shù)后當(dāng)天起，即給予辛伐他汀25mg/kg/d，自由進(jìn)食水不限。此后每隔24h投予相同劑量的辛伐他汀，直至術(shù)后1周、4周。雙側(cè)結(jié)扎組術(shù)后除不予藥物處理外，余同辛伐他汀組。每組術(shù)后1周、4周分別取6只兔子基底動(dòng)脈行病理切片觀察，另外每組12只分別行PCR和Western blot檢測(cè)。另取6只假手術(shù)組兔子基底動(dòng)脈作為對(duì)照檢測(cè)eNOS、VCAM-1和MMP-9mRNA表達(dá)。 結(jié)果：二組在1W和4W時(shí)基底動(dòng)脈分叉部動(dòng)脈瘤樣改變?cè)诎l(fā)生數(shù)量上無(wú)顯著差異，但在辛伐他汀干預(yù)組基底動(dòng)脈分叉部?jī)?nèi)彈力膜薄弱長(zhǎng)度縮短（P＜0.05）、中層平滑肌變薄程度輕（P＜0.05）和瘤體最長(zhǎng)徑較雙側(cè)結(jié)扎組縮小，具有顯著統(tǒng)計(jì)學(xué)差異（P＜0.05）。辛伐他汀組基底動(dòng)脈分叉部樣本中，VCAM-1和MMP-9mRNA在術(shù)后1W和術(shù)后4W其表達(dá)均下降，而eNOS mRNA表達(dá)上升，且與雙側(cè)結(jié)扎組相同時(shí)間點(diǎn)比較具有顯著統(tǒng)計(jì)學(xué)意義(P＜0.05)。KLF2Western-blot蛋白半定量結(jié)果發(fā)現(xiàn)： KLF2的表達(dá)升高，在4W時(shí)升高更為明顯，與雙側(cè)結(jié)扎組比較，其表達(dá)具有顯者的統(tǒng)計(jì)學(xué)差異(P＜0.05) 結(jié)論：辛伐他汀在25mg/kg/d的劑量，能顯著上調(diào)雙側(cè)結(jié)扎組KLF2的表達(dá)。KLF2/eNOS、VCAM-1、MMP-9途徑參與了顱內(nèi)動(dòng)脈瘤發(fā)生。辛伐他汀能夠減輕基底動(dòng)脈分叉部動(dòng)脈瘤樣凸起的病理改變。
[Abstract]:The expression of the first part of klF2 in the animal model of aneurysm induced by hemodynamics

Objective : To observe the expression of kulele factor 2 and its correlation with the changes of arterial blood flow velocity in aneurysm model induced by hemodynamics induced by different methods .

Methods : 144 New Zealand white rabbits were randomly divided into three groups : sham operation group , unilateral carotid artery ligation group and bilateral common carotid artery ligation group .

Results : The results showed that there was no significant difference between the two groups ( P > 0.05 ) . The results showed that there was no significant difference between the two groups ( P > 0.05 ) .

Conclusion : 1 . The hemodynamic changes induced by simple hemodynamics induced by the ligation of bilateral common carotid artery were the most obvious and more suitable for the animal model of aneurysm research .

2 . There was agreement between the change of expression and the change of blood flow velocity , and the correlation analysis suggested that both were positively correlated , suggesting that the expression was regulated by hemodynamics .

The expression of miR - 92a upstream regulatory factor miR - 92a in the animal model induced by hemodynamics

Objective : To investigate the expression of miRNA - 92a in each group of basilar artery bifurcation by means of RT - PCR , and to explore its regulation and control effect .

Methods : 72 male New Zealand white rabbits were randomly divided into two groups : sham operation group , unilateral common carotid artery ligation group and bilateral common carotid artery ligation group . Each group was divided into 1 week , 2 week , 3 week and 4 week group . The expression of miRNA - 92a was detected by RT - PCR at each time point .

Results : The expression level of mir - 92a mRNA in bilateral ligation group and bilateral ligation group was significantly lower than that in sham operation group ( P < 0.05 ) . Compared with sham operation group and unilateral ligation group , the expression level of mir - 92a was significantly negative ( r = - 0.768 , P < 0.01 ) , but also negative correlation with the basal arterial blood flow velocity correlation analysis ( r = - 0.709 , P < 0.01 ) .

Conclusion : The expression of miRNA - 92a mRNA decreased with the increase of blood flow velocity and negatively correlated with the changes of blood flow velocity .
The expression of miRNA - 92a mRNA was regulated by hemodynamics .

Effect of the third part of simvastatin on the expression of klF2 and downstream factors in the aneurysm model

Objective : To investigate the mechanism of MMP - 1 and MMP - 9 in the pathogenesis of aneurysm and the effect of simvastatin on aneurysm growth .

Methods : 72 New Zealand white rabbits were randomly divided into two groups : simvastatin intervention group and bilateral common carotid artery ligation group . Each group was divided into 1 week and 4 weeks after operation .

Results : Compared with bilateral ligation group , the expression of VCAM - 1 and MMP - 9 mRNA decreased significantly ( P & lt ; 0.05 ) , and the expression of VCAM - 1 and MMP - 9 mRNA was significantly higher than that in bilateral ligation group ( P & lt ; 0.05 ) .

Conclusion : The dose of simvastatin in 25 mg / kg / d can significantly increase the expression of klF2 in the bilateral ligation group , which is involved in the occurrence of intracranial aneurysms .
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

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