兔急性心肌梗死后室壁瘤形成过程心肌力学及MMP-9变化的实验研究
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摘要
目的：本研究通过建立兔急性心肌梗死后室壁瘤(LVA)形成动物模型，使用实时三维超声心动图和二维超声斑点追踪技术观察兔急性心肌梗死后室壁瘤形成过程中室壁瘤的形态学变化及心肌力学演变特点，并观察金属蛋白酶-9(MMP-9)在室壁瘤形成过程中的作用，旨在探讨兔急性心肌梗死后室壁瘤形成的结构和力学演变规律，为临床早期诊断、早期干预治疗室壁瘤提供一定的理论依据。方法：第一部分：新西兰大白兔80只，分为4组，每组20只。根据不同开胸方式和结扎不同冠脉建立兔急性心肌梗死后室壁瘤形成动物模型。A组采用剪开正中胸骨开胸，结扎兔左前降支中段；B组采用剪开正中胸骨开胸，结扎兔左前降支和左旋支中段；C组采用左侧胸腔3～4肋间隙开胸，结扎兔左前降支中段；D组采用左侧胸腔3～4肋间隙开胸，结扎兔左前降支及左旋支中段。饲养4周后，比较不同开胸方式和结扎不同冠脉建立兔室壁瘤模型的成功率、存活率及室壁瘤形成特点。第二部分：20只新西兰白兔建立室壁瘤形成动物模型，使用实时三维超声心动图和二维超声斑点追踪技术于模型建立术前、术后1d、2d、3d、1w、2w、3w、4w进行检查。4周后，将存活至实验终点且有室壁瘤形成的实验动物纳入研究。实时三维超声心动图观察不同时间段兔室壁瘤形成过程中室壁瘤的大小、形态、形成时间及心功能的变化。二维超声斑点追踪技术观察不同时间段兔室壁瘤形成过程中径向应变率和圆周应变率收缩期与舒张期的变化特点及其与心功能的关系。第三部分：80只新西兰白兔分为正常对照组与实验组，实验组建立室壁瘤模型，并于术前、术后1d、2d、3d、1w、2w、3w、4w进行实时三维超声心动图检查，4周后，将存活至实验终点且有室壁瘤形成的实验动物纳入室壁瘤组，无室壁瘤形成的动物纳入心肌梗死组。使用血清学、免疫组化、荧光定量PCR技术、weston-blot蛋白技术观察MMP-9在室壁瘤形成过程中的表达情况，并与心肌梗死组比较。结果：第一部分：（1）动物存活率比较， A组、C组动物存活率较高，分别为90%、95%。B组、D组存活率较低，存活率分别为65%、70%。A组、C组存活率与B组、D组比较，差异有统计学意义（P＜0.05）。A组与C组、B组与D组动物存活率比较，差异无统计学意义（P＞0.05）。（2）各组模型成功率比较， B组、D组室壁瘤形成率较高，分别为84%、86%。A组、C组成功率较低，分别为50%、53%。A组、C组与B组、D组成功率比较，差异有统计学意义（P＜0.05）。A组与C组、B组与D组比较，差异无统计学意义（P＞0.05）。（3）超声心动图参数比较，B组、D组的LVEDV、LVESV、LVA容积较A组、C组增加显著（P＜0.05），LVEF下降明显（P＜0.05），差异有统计学意义。第二部分：（1）术后各时间段存活实验动物中，术后1d无室壁瘤形成，术后2d室壁瘤开始形成，术后2d与3w之间室壁瘤形成例数呈增加趋势。3W之后无新的室壁瘤形成。（2）与术前比较，术后各时间段LVEDV、LVESV、LVAV呈增加趋势（P＜0.05），术后3w后趋于稳定。与术前比较，术后LVEF呈下降趋势（P＜0.05），3w后下降趋于稳定。LVEDV、LVAV、LVAV/LVEDVD与LVEF均有相关性（r=0.232、0.778、0.911）。其中LVAV/LVEDV与EF值相关性较好，根据直线相关方程发现LVAV/LVEDV每增加1%，LVEF值下降1.1%。判定LVEF小于50%的LVAV/LVEDV临界值为16%。（3）各组间SrR、SrC比较。与术前比较，术后各时间段各节段SrR-S、SrR-E、SrR-A、SrC-S、 SrC-E及SrC-A均明显下降（P＜0.05）。术后各组间比较，术后2d、3d、1w、2w、3w、4w组各节段SrR-S、SrR-E、SrR-A、SrC-S、 SrC-E及SrC-A较术后1d下降显著（P＜0.05），术后3w SrR-S、SrR-E、SrR-A、SrC-S、 SrC-E及SrC-A与术后4w组比较，差异无统计学意义（P＞0.05）。术后同一时间段各节段间比较，前壁、侧壁SrR-S、SrC-S较其他节段下降显著（P＜0.05），舒张期各节段间SrR-E、SrR-A、、SrC-E及SrC-A差别无统计学意义（P＞0.05）。各节段SrC-S、SrR-S与LVEF均有较好的相关性，其中前壁、侧壁SrC-S、SrR-S与LVEF相关性较好。（4）室壁瘤形成后各节段应变率下降百分比比较，收缩期各节段间比较，球性检验显示各节段间径向应变率、圆周应变率下降百分比差异有统计学意义（F=6.03、6.08，P＜0.05）其中前壁、侧壁下降明显（P＜0.05）。舒张早期、舒张晚期各节段径向应变率及圆周应变率下降百分比之间比较，差异无统计学意义（P＞0.05）。ROC曲线显示，前壁、侧壁收缩期圆周应变率下降百分比曲线下面积分别为0.889、0.861，前壁、侧壁收缩期径向应变率下降百分比曲线下面积分别为0.917、0.887，以应变率下降百分比值60%为临界点预测室壁瘤的形成，前壁、侧壁圆周应变率下降百分比特异性分别为73.6%、78.2%，灵敏性83.3%、83.3%。前壁、侧壁径向应变率下降百分比特异性分别为69.7%、83.3%，灵敏性83.3%、67.7%。第三部分：（1）与术前比较，室壁瘤组各时间段MMP-9血清浓度、阳性细胞表达、mRNA CT、蛋白半定量值均增高，术后3d达到高峰，3d后开始下降，至术后4w MMP-9仍然高于术前，差异有统计学意义（P＜0.05）。心肌梗死组术后MMP-9血清浓度、阳性细胞表达、mRNA CT、蛋白半定量值变化趋势与室壁瘤组基本一致，但室壁瘤组术后1w后下降较心肌梗死组缓慢（P＜0.05）（2）MMP-9与室壁瘤容积及LVEF有较好的相关性（r=0.65，0.78）。结论：（1）结扎不同冠状动脉对室壁瘤模型的动物存活率和室壁瘤形成率有着直接的影响和决定作用，结扎左前降支及左旋支中段模型制作成功率较高，且形成容积较为恒定的室壁瘤，使用实时三维超声心动图可以较为直观、客观的评价室壁瘤动物模型。（2）急性心肌梗死后2d室壁瘤开始形成，3w后室壁瘤基本形成，室壁瘤容积随时间呈增大趋势，室壁瘤容积与左室舒张末期容积之比可作为室壁瘤形成后评估心功能的重要参考指标。（3）室壁瘤形成后加重了各节段心肌径向及圆周应变率的下降，尤以前壁及侧壁下降明显。前壁及侧壁收缩期心肌应变率下降百分比可作为预测室壁瘤和评估心功能新的参数，二维超声斑点追踪成像技术对于研究室壁瘤形成过程中的心肌力学演变能够提供有效的量化检测手段。（4）MMP-9参与了室壁瘤的形成过程并对室壁瘤容积和心功能变化有着较为重要的影响。MMP-9可作为判断室壁瘤容积及心功能的一个新的有价值指标。
Objective: The research aims to invistgate the changes and evolution of morphologyby real time three dimensional echocardiography (RT-3DE)and myocardial mechanicsusing the two dimensional speckle tracking imaging technology(2D-STI) during leftventricular aneurysm (LVA) formation after acute myocardial infarction (AMI) in rabbits.And explore the effect of matrix metalloproteinase-9(MMP-9) on LVA formation afterAMI from the serology and molecular biology, which provide the theoretical basis forclinical early diagnosis, early treatment LVA and evaluating cardiac function. Methods:Part one:This part is about the evaluating animal livability and the formation rate of LVAafter AMI.80New Zealand rabbits were divide into four group according to the differentway of thoracotomy and ligating the different coronary artery. Group A: A rabbit model ofLVA was prepared in20New Zealand rabbits by ligating the middle segment of the leftanterior descending artery and thoracotomy by cuting the middle sternum. Group B: Arabbit model of LVA was prepared in20New Zealand rabbits by ligating the middlesegment of the left anterior descending artery and the left circumflex artery andthoracotomy by cuting the middle sternum. Group C: A rabbit model of LVA was preparedin20New Zealand rabbits by ligating the middle segment of the left anterior descendingartery and thoracotomy was performed through the left fourth intercostal space. Group D:A rabbit model of LVA was prepared in20New Zealand rabbits by ligating the middlesegment of the left anterior descending artery and the left circumflex artery andthoracotomy was performed through the left fourth intercostal space. At4weeks after theprocedure,the LVA characteristics, animal livability and the formation rate of LVA were compared with four group. Part two: A rabbit of LVA was established in20New Zealandrabbits by ligating the middle segment of the left anterior descending artery and the leftcircumflex artery and thoracotomy was performed through the left fourth intercostal space.RT-3DE and2D-STI were performed at preoperative and postoperative1d,2d,3d,1w,2w,3w,4w. At4weeks later, the survived animals which haved formed LVA wereenrolled into this study. RT-3DE was uses to observe the LVA volume, shape, formatingtime and the changes of heart function at different time.2D-STI was performed tomeasure the changes of radial strain rate (SrR) and circumferential strain rate (SrC) atsystolic and diastolic at different time. Part three: A rabbit of LVA was established in20New Zealand rabbits by ligating the middle segment of the left anterior descending arteryand the left circumflex artery and thoracotomy was performed through the left fourthintercostal space. RT-3DE was performed at preoperative and postoperative1d,2d,3d,1w,2w,3w and4w. At4weeks later, the survived animals which had formed LVA wereenrolled into this study. The MMP-9serum concentration was measured by the doubleantibody sandwich ABC-ELISA method and expression of MMP-9was detected byimmunohistochemistry,while mRNA and protein were detected by RT-PCR and WesternBlot at different time phase, respectively. Results: Part one:(1) The animal livability ingroup A, B, C, D was90%,65%,95%and70%respectively. Compared with group B andD, there were significantly higher in group A and C (P<0.05).(2) The formation rate ofLVA after AMI in group A, B, C and D was50%,84%,53%and86%, respectively.Compared with group B and D, there were significantly higher in group A and C (P<0.05).(3) Compared with group A and C, LVEDV, LVESV, and LVAV were significantlyenlarged in group B and D, while LVEF was significantly decreased, there were statisticsdifference(P<0.05). Part two:(1) In the survived animals, the LVA come into being atpostoperative2d. There was an increase trend in the amount of LVA formation frompostoperative2d to postoperative3w. While the LVEDV, LVESV and LVAV showed anincrease trend from postoperative2d to postoperative3w (P<0.05), however there was adecreasing trend in LVEF from postoperative2d to postoperative3w (P<0.05). Therewere no statistics difference between postoperative3w and postoperative4w (P>0.05).Moreover, there were high correlations between LVEF and LVAV, LVA volume/LVEDV(r=0.778,0.911, respectively, all p<0.05). Of note, had the tightest inverse relationshipwith LVEF (r=-0.911, p <0.01). Specifically, LVAV/LVEDV>16%corresponded to LVEF<50%, and LVEF decreased1.1%while LVAA/LVEDV increased1%.(2) Compared withpreoperative, there were significant decrease in the SrR-S, SrR-E, SrR-A,SrC-S, SrC-E and SrC-A at postoperative1d,2d,3d,1w,2w,3w and4w(P<0.05). Compared withpostoperative1d, there were significant decrease in the SrR-S, SrR-E, SrR-A,SrC-S,SrC-E and SrC-A at postoperative2d,3d,1w,2w,3w and4w(P<0.05), while Therewere no statistics difference between postoperative3w and postoperative4w (P>0.05).SrC-Santerior wall, SrR-Santerior wall, SrC-Slateral wall, and SrR-S lateral wall weresignificant decrease than other segment at the same time postoperative (P<0.05), andSrC-Santerior wall, SrR-Santerior wall, SrC-Slateral wall, and SrR-S lateral wall hadhigh correlations with LVEF((r=0.895,n0.887,0.890,0.891, respectively, all p <0.05).(4)Compared with the each segment in the strain rate decrease percentage at systole, therewere significant decrease at anterior and lateral wall (P<0.05). while There were nostatistics difference at each segment at diastole (P>0.05).The ROC curve analysis showedthat when the strain rate decrease percentage was60%as the critical point, the strain ratedecrease percentage of SrR-Santeriorwall, SrR-Slateralwall, SrC-Santeriorwall andSrC-Slateralwall had a sensitivity of83.30%,84.40%,83.30%and67.70%and aspecificity was73.60%,78.20%,69.70%and83.30%for prognosis ventricularneurysm,repectively. Part three: Compared with preoperative, there were an significantincrease in the serum concentration of MMP-9, expression of MMP-9positive cell,mRNA CT value of MMP-9and Protein quantitative value of MMP-9at postoperative1d2d,3d (P<0.05), and arrived at the peak at postoperative3d, decreased frompostoperative3d to postoperative4w, respectively, and till postoperative4w,there weremore high level than preoperative (all p <0.05). In AMI group, there was the same changetrend with LVA group, however the mangitute of decrease was larger than the mandututein LVA group(p <0.05).(2)There were good correlations between MMP-9and LVAvolumenand LVEF (r=0.65,0.78, respectively, all p<0.05)..Conclusion: The conclusionof the research can be drawn from the following four aspects,(1) It maybe can play theimportant role in establishment of animal model of LVA after AMI that differentcoronary artery is ligated. Real-time three-dimensional echocardiography can be as a newmethods to accurately measure the LVA volume and evaluate the LVA morphology.(2)LVA comes to being at postoperative2d and completely forms at postoperative3w. TheLVA volume increasingly enlarges from postoperative2d to postoperative3w. Theparameter of LVA volume/LVEDV can be as a new indictor for evaluating the cardiacfunction.(3) The myocardial SrR and SrC significantly reduce after VA formation,especially in the anterior and lateral segments.The strain rate decrease percentage in theanterior and lateral segments at systole could be as an important indicator to predict the formation of VA after myocardial infarction.(4) MMP-9all along play a key role in theLVR after AMI from postoperative1d to postoperative4w, and has a close influence onLVA formation after AMI, and has a high correlation with LVA volume and LVEF, whichmaybe as a new indictor to predict the LVA volume and LVEF.
Objective: The research aims to invistgate the changes and evolution of morphologyby real time three dimensional echocardiography (RT-3DE)and myocardial mechanicsusing the two dimensional speckle tracking imaging technology(2D-STI) during leftventricular aneurysm (LVA) formation after acute myocardial infarction (AMI) in rabbits.And explore the effect of matrix metalloproteinase-9(MMP-9) on LVA formation afterAMI from the serology and molecular biology, which provide the theoretical basis forclinical early diagnosis, early treatment LVA and evaluating cardiac function. Methods:Part one:This part is about the evaluating animal livability and the formation rate of LVAafter AMI.80New Zealand rabbits were divide into four group according to the differentway of thoracotomy and ligating the different coronary artery. Group A: A rabbit model ofLVA was prepared in20New Zealand rabbits by ligating the middle segment of the leftanterior descending artery and thoracotomy by cuting the middle sternum. Group B: Arabbit model of LVA was prepared in20New Zealand rabbits by ligating the middlesegment of the left anterior descending artery and the left circumflex artery andthoracotomy by cuting the middle sternum. Group C: A rabbit model of LVA was preparedin20New Zealand rabbits by ligating the middle segment of the left anterior descendingartery and thoracotomy was performed through the left fourth intercostal space. Group D:A rabbit model of LVA was prepared in20New Zealand rabbits by ligating the middlesegment of the left anterior descending artery and the left circumflex artery andthoracotomy was performed through the left fourth intercostal space. At4weeks after theprocedure,the LVA characteristics, animal livability and the formation rate of LVA were compared with four group. Part two: A rabbit of LVA was established in20New Zealandrabbits by ligating the middle segment of the left anterior descending artery and the leftcircumflex artery and thoracotomy was performed through the left fourth intercostal space.RT-3DE and2D-STI were performed at preoperative and postoperative1d,2d,3d,1w,2w,3w,4w. At4weeks later, the survived animals which haved formed LVA wereenrolled into this study. RT-3DE was uses to observe the LVA volume, shape, formatingtime and the changes of heart function at different time.2D-STI was performed tomeasure the changes of radial strain rate (SrR) and circumferential strain rate (SrC) atsystolic and diastolic at different time. Part three: A rabbit of LVA was established in20New Zealand rabbits by ligating the middle segment of the left anterior descending arteryand the left circumflex artery and thoracotomy was performed through the left fourthintercostal space. RT-3DE was performed at preoperative and postoperative1d,2d,3d,1w,2w,3w and4w. At4weeks later, the survived animals which had formed LVA wereenrolled into this study. The MMP-9serum concentration was measured by the doubleantibody sandwich ABC-ELISA method and expression of MMP-9was detected byimmunohistochemistry,while mRNA and protein were detected by RT-PCR and WesternBlot at different time phase, respectively. Results: Part one:(1) The animal livability ingroup A, B, C, D was90%,65%,95%and70%respectively. Compared with group B andD, there were significantly higher in group A and C (P<0.05).(2) The formation rate ofLVA after AMI in group A, B, C and D was50%,84%,53%and86%, respectively.Compared with group B and D, there were significantly higher in group A and C (P<0.05).(3) Compared with group A and C, LVEDV, LVESV, and LVAV were significantlyenlarged in group B and D, while LVEF was significantly decreased, there were statisticsdifference(P<0.05). Part two:(1) In the survived animals, the LVA come into being atpostoperative2d. There was an increase trend in the amount of LVA formation frompostoperative2d to postoperative3w. While the LVEDV, LVESV and LVAV showed anincrease trend from postoperative2d to postoperative3w (P<0.05), however there was adecreasing trend in LVEF from postoperative2d to postoperative3w (P<0.05). Therewere no statistics difference between postoperative3w and postoperative4w (P>0.05).Moreover, there were high correlations between LVEF and LVAV, LVA volume/LVEDV(r=0.778,0.911, respectively, all p<0.05). Of note, had the tightest inverse relationshipwith LVEF (r=-0.911, p <0.01). Specifically, LVAV/LVEDV>16%corresponded to LVEF<50%, and LVEF decreased1.1%while LVAA/LVEDV increased1%.(2) Compared withpreoperative, there were significant decrease in the SrR-S, SrR-E, SrR-A,SrC-S, SrC-E and SrC-A at postoperative1d,2d,3d,1w,2w,3w and4w(P<0.05). Compared withpostoperative1d, there were significant decrease in the SrR-S, SrR-E, SrR-A,SrC-S,SrC-E and SrC-A at postoperative2d,3d,1w,2w,3w and4w(P<0.05), while Therewere no statistics difference between postoperative3w and postoperative4w (P>0.05).SrC-Santerior wall, SrR-Santerior wall, SrC-Slateral wall, and SrR-S lateral wall weresignificant decrease than other segment at the same time postoperative (P<0.05), andSrC-Santerior wall, SrR-Santerior wall, SrC-Slateral wall, and SrR-S lateral wall hadhigh correlations with LVEF((r=0.895,n0.887,0.890,0.891, respectively, all p <0.05).(4)Compared with the each segment in the strain rate decrease percentage at systole, therewere significant decrease at anterior and lateral wall (P<0.05). while There were nostatistics difference at each segment at diastole (P>0.05).The ROC curve analysis showedthat when the strain rate decrease percentage was60%as the critical point, the strain ratedecrease percentage of SrR-Santeriorwall, SrR-Slateralwall, SrC-Santeriorwall andSrC-Slateralwall had a sensitivity of83.30%,84.40%,83.30%and67.70%and aspecificity was73.60%,78.20%,69.70%and83.30%for prognosis ventricularneurysm,repectively. Part three: Compared with preoperative, there were an significantincrease in the serum concentration of MMP-9, expression of MMP-9positive cell,mRNA CT value of MMP-9and Protein quantitative value of MMP-9at postoperative1d2d,3d (P<0.05), and arrived at the peak at postoperative3d, decreased frompostoperative3d to postoperative4w, respectively, and till postoperative4w,there weremore high level than preoperative (all p <0.05). In AMI group, there was the same changetrend with LVA group, however the mangitute of decrease was larger than the mandututein LVA group(p <0.05).(2)There were good correlations between MMP-9and LVAvolumenand LVEF (r=0.65,0.78, respectively, all p<0.05)..Conclusion: The conclusionof the research can be drawn from the following four aspects,(1) It maybe can play theimportant role in establishment of animal model of LVA after AMI that differentcoronary artery is ligated. Real-time three-dimensional echocardiography can be as a newmethods to accurately measure the LVA volume and evaluate the LVA morphology.(2)LVA comes to being at postoperative2d and completely forms at postoperative3w. TheLVA volume increasingly enlarges from postoperative2d to postoperative3w. Theparameter of LVA volume/LVEDV can be as a new indictor for evaluating the cardiacfunction.(3) The myocardial SrR and SrC significantly reduce after VA formation,especially in the anterior and lateral segments.The strain rate decrease percentage in theanterior and lateral segments at systole could be as an important indicator to predict the formation of VA after myocardial infarction.(4) MMP-9all along play a key role in theLVR after AMI from postoperative1d to postoperative4w, and has a close influence onLVA formation after AMI, and has a high correlation with LVA volume and LVEF, whichmaybe as a new indictor to predict the LVA volume and LVEF.
引文
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