克鲁佩尔因子2及其上下游通路在血流动力学诱导的动脉瘤生长中的作用

发布时间：2018-06-07 01:09

本文选题：克鲁佩尔因子2 + 血流动力学　；参考：《第二军医大学》2014年博士论文

【摘要】：第一部分KLF2在血流动力学诱导的动脉瘤动物模型中的表达目的：观察不同方法构建的血流动力学诱导动脉瘤模型中克鲁佩尔因子2（KLF2）的表达及与基底动脉血流速度变化的相关性。方法：雄性新西兰白兔144只，随机分成三组：假手术组、单侧颈总动脉结扎组、双侧颈总动脉结扎组，每组48只，每组各分为术后1周、2周、3周和4周组，每组12只。假手术组仅切开颈部暴露颈总动脉不结扎，单侧结扎组统一结扎右侧颈总动脉，双侧结扎组进行双侧颈总动脉结扎。各组分别在术后1周、2周、3周、4周行经颅多普勒超声测量基底动脉血流速度。然后在各时间点处死动物，每组每个时间点取6只基底动脉分叉部进行HE染色、EVG染色、Masson染色、KLF2免疫组化染色等病理检测，另外6只基底动脉行KLF2western blot蛋白分析。结果：各组基底动脉平均血流速度测量结果，在假手术组术后不同时间点变化不明显，单侧颈总动脉结扎组术后血流速度较假手术组增加，但统计学结果提示无显著差异（P＞.05）。双侧颈总动脉结扎组流速明显快于假手术组和单侧颈总动脉结扎组，有显著统计学意义（p＜0.05）。病理学检查结果提示按照动脉瘤评价标准，假手术组基底动脉尖端管腔平滑，内皮细胞完整，内弹力膜完整，无断裂和变薄，全组无动脉瘤形成。右侧结扎组中24只动物模型中有1只基底动脉尖端略向外膨出，但膨出程度比较低。双侧结扎组中有12只向外凸出的动脉瘤样突起，统计分析表明三组间具有显著统计学差异（p＜0.05）。KLF2western blot定量分析提示假手术组基底动脉的KLF2蛋白表达随时间变化无明显变化，而单侧结扎组蛋白表达随时间变化有所增加，但增加较少。双侧结扎组KLF2蛋白表达在术后1W起即有显著增加，随后一直维持在较高水平，统计结果显示与假手术组和单侧结扎组相比具有统计学意义(P＜0.05)。KLF2蛋白表达与血流速度变化相关分析表明二者具有显著正相关性（r=0.845， P＜0.05）。结论：1、采用兔双侧颈总动脉结扎法建立的单纯血流动力学诱导的动脉瘤模型引起的血流动力学变化最为明显，更适合做为动脉瘤研究的动物模型。 2、KLF2的表达变化与血流速度变化具有一致性，相关分析提示二者明显正相关，，提示其表达受血流动力学调控。第二部分KLF2上游调控因子miR-92a在血流动力学诱导动脉瘤动物模型中的表达目的：利用不同方法构建的血流动力学诱导的动脉瘤模型，采用RT-PCR法检测各组基底动脉分叉部miRNA-92a的表达差异，探讨其对KLF2的表达的调控作用。方法：雄性新西兰白兔72只，随机分成假手术组、单侧颈总动脉结扎组、双侧颈总动脉结扎组各24只，每组各分为术后1周、2周、3周和4周组，每组6只。然后在各时间点处死动物，取基底动脉分叉部进行RT-PCR检测miRNA-92a的表达差异。其表达结果与基底动脉血流速度进行相关分析。结果：单侧结扎组和双侧结扎组基底动脉分叉部mir-92a mRNA表达水平较假手术组明显降低。双侧结扎组降低最为明显，与假手术组和单侧结扎组相比具有显著统计学差异(P＜0.05)。mir-92a的mRNA表达水平与KLF2蛋白表达相关分析表明二者显著呈负相关（r=-0.768, P＜0.01），而与基底动脉血流速度相关分析提示二者亦呈负相关（r=-0.709, P＜0.01）。结论： miRNA-92a mRNA表达随血流速度的增加而表达下降，且与血流速度的变化具有负相关性；说明miRNA-92a mRNA表达受血流动力学调控。miRNA-92a表达与KLF2表达趋势相反，二者呈显著负相关。第三部分辛伐他汀对KLF2及下游因子在动脉瘤模型中的表达影响目的：探讨KLF2/eNOS、VCAM-1、MMP-9通路在动脉瘤发生中的作用机制及辛伐汀抑制动脉瘤生长的作用。方法：新西兰白兔72只，随机分成二组：双侧颈总动脉结扎后辛伐他汀干预组、双侧颈总动脉结扎组各36只，每组各分为术后1周、4周，每组18只。辛伐他汀组自动物双侧颈总动脉结扎术后当天起，即给予辛伐他汀25mg/kg/d，自由进食水不限。此后每隔24h投予相同剂量的辛伐他汀，直至术后1周、4周。双侧结扎组术后除不予药物处理外，余同辛伐他汀组。每组术后1周、4周分别取6只兔子基底动脉行病理切片观察，另外每组12只分别行PCR和Western blot检测。另取6只假手术组兔子基底动脉作为对照检测eNOS、VCAM-1和MMP-9mRNA表达。结果：二组在1W和4W时基底动脉分叉部动脉瘤样改变在发生数量上无显著差异，但在辛伐他汀干预组基底动脉分叉部内弹力膜薄弱长度缩短（P＜0.05）、中层平滑肌变薄程度轻（P＜0.05）和瘤体最长径较双侧结扎组缩小，具有显著统计学差异（P＜0.05）。辛伐他汀组基底动脉分叉部样本中，VCAM-1和MMP-9mRNA在术后1W和术后4W其表达均下降，而eNOS mRNA表达上升，且与双侧结扎组相同时间点比较具有显著统计学意义(P＜0.05)。KLF2Western-blot蛋白半定量结果发现： KLF2的表达升高，在4W时升高更为明显，与双侧结扎组比较，其表达具有显者的统计学差异(P＜0.05) 结论：辛伐他汀在25mg/kg/d的剂量，能显著上调双侧结扎组KLF2的表达。KLF2/eNOS、VCAM-1、MMP-9途径参与了颅内动脉瘤发生。辛伐他汀能够减轻基底动脉分叉部动脉瘤样凸起的病理改变。
[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 .
【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743

【共引文献】