【作者】 叶明；

【导师】 贾兵；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 博士

【摘要】 概述：随着体外循环手术的低龄化和复杂化,术后心力衰竭的发生率明显增加,因此早期诊断和及时治疗至关重要。心力衰竭的发生机制主要是神经内分泌系统的激活和心室重构。心室重构包括心肌细胞和细胞外基质的变化,后者是心室重构的重要原因,又称为心肌基质重构。基质金属蛋白酶是一组能特异地降解细胞外基质成分的Zn2+依赖的酶家族,在组织重构中起重要作用。基质金属蛋白酶(Matrix Metalloproteinase, MMPs)与基质金属蛋白酶抑制剂(Tissue Inhibitor of Metalloproteinase, TIMPs)是胶原代谢的主要调节物质,两者相互作用共同维持着心血管基质的分解与重构。在冠心病、高血压的病人中存在MMPs活性增高,TIMPs表达减少,MMPs与TIMPs平衡失调,导致细胞外基质合成和降解失衡,心室扩大,心功能下降。本研究为了了解先天性心脏病MMPs与TIMPs在血浆和心肌组织中的表达水平,比较充血类和缺血类先心围术期MMPs与TIMPs表达的差异,并进一步应用血管紧张素转换酶抑制剂(Angiotension Converting Enzyme Inhibitors, ACEI)治疗肺充血性先心,了解用药前后MMPs与TIMPs表达的变化。第一部分基质金属蛋白酶及其抑制剂在先天性心脏病中表达的研究目的：了解先天性心脏病患儿血浆MMP-3、MMP-9、TIMP-1的浓度,比较肺充血性先心与缺血类先心的差异,并探讨其可能的机制。方法：选择2009年1月～2009年8月心胸外科手术患儿55例,分为三组,漏斗胸10例为正常对照组,室间隔缺损患儿25例为肺充血组,法洛四联症患儿20例为肺缺血组。所有患儿术前行超声心动图检查,测左室射血分数(LVEF)、左心室收缩及舒张末直径和容积(LVDs、LVDd、LVESV、LVEDV)。术前抽血,应用ELISA法检测血浆MMP-3,-9及TIMP-1浓度。结果：超声心动图检查：1.肺充血组较正常组LVEDV、LVESV、LVDd、LVDs均增大,p<0.01。2.肺缺血组较正常组比较各参数均减小,但是p>0.05,仅LVEDV减小有统计学意义。3.肺充血组和缺血组比较除LVEF无差异外,LVEDV、LVESV、LVDd、LVDs均增大,p<0.01,有显著统计学意义。实验室检测：ELISA1.MMP-3：肺充血组较缺血组及对照组升高,p<0.01,有显著统计学意义；缺血组与对照组比较无差异。2. MMP-9：肺充血组与肺缺血组均高于正常组,p<0.05,有统计学意义,且以肺充血组升高更为显著。3.TIMP-1：肺充血组与肺缺血组均低于正常组,p<0.05,有统计学意义,而以肺缺血组降低更为显著。结论：1.根据超声心动图检查结果与正常对照组比较,说明先天性心脏病患儿存在不同程度的心功能不全。2.先天性心脏病的患儿存在血浆MMP-3,-9及TIMP-1浓度的改变,与对照组比较MMP-3、-9均升高,而TIMP-1降低。3.充血性先心和缺血类先心相比,血浆MMP-3、-9、TIMP-1浓度均升高,提示肺充血性先心的心功能改变可能较缺血类先心更为显著。第二部分肌钙蛋白与基质金属蛋白酶及抑制剂在先天性心脏病围术期的表达及机制目的：了解肺充血性先心与缺血类先心心肌组织中MMP-3、-9、TIMP-1 mRNA的表达及心肌组织胶原含量和分布的差异,比较肺充血性先心和缺血类先心在体外循环手术前后血浆MMP-3、-9、TIMP-1浓度,并探讨其可能的机制。同时测定肌钙蛋白(cTnI)在体外循环前后的变化,比较cTnⅠ与MMPs/TIMPs及心功能的相关性。方法：选取2009年1月～2009年8月在我院行体外循环手术治疗的先天性心脏病患儿共45例,室间隔缺损25例为肺充血组,法洛四联症20例为肺缺血组。所有患儿于术前、术后一周行超声心动图检查,测LVEF、LVDs、LVDd、LVESV、LVEDV。分别于术前、体外循环术后即刻、术后6h、24h、3d抽血测血浆MMP-3,-9及TIMP-1浓度以及测cTnI的变化。术中取右室异常肌束应用RT-PCR方法检测MMP-3,-9及TIMP-1 mRNA表达,应用羟脯氨酸消化法测心肌羟脯氨酸的含量,并进一步计算心肌胶原含量。取右室异常肌束应用免疫组化的方法,了解Ⅰ、Ⅲ型胶原的分布。结果：超声心动图检查：1.肺充血组术前、术后比较除LVEF无变化外,LVEDV、LVESV、LVDd、LVDs均较术前减小,p<0.01,有显著统计学意义。2.肺缺血组术前、术后比较LVEF、LVEDV、LVESV、LVDd、LVDs均有所增加,但除LVDs外(p<0.05),其余测值均无统计学意义。3.术后肺充血组与缺血组之间比较各参数均无统计学差异,p>0.05。实验室检测：ELISA:1.术前及术后各时点肺充血组血浆MMP-3、-9、TIMP-1浓度均高于肺缺血组,p<0.05,且有统计学意义。肺充血组cTnⅠ浓度在术前高于缺血组,但术后各时点均低于缺血组,p<0.05,有统计学意义。2.同一组内CPB前后不同时点比较：1)MMP-3：于CPB术后升高,术后6h达最高,然后逐渐降低,至术后3天降至低于术前水平。2)MMP-9：于CPB术后即刻显著升高,术后6h达最高,以后逐渐下降,至术后3天时降至低于术前水平。3)TIMP-1：两组均表现为逐渐升高,肺充血组于术后3天达到最高,而肺缺血组在术后3天时呈逐渐下降的趋势。4)cTnI：两组均表现为术后即刻显著升高,以后逐渐降低,至术后3天肺缺血组仍高于术前。RT-PCR:1.肺充血组心肌组织中MMP-3、-9、TIMP-1 mRNA表达均高于肺缺血组,p<0.05,有统计学意义。2.肺充血组中MMP-3 mRNA的含量最低,MMP-9与TIMP-1的含量相仿。3.肺缺血组中MMP-3 mRNA的含量最低,TIMP-1 mRNA的含量最高。羟脯氨酸消化法：肺充血组心肌组织羟脯氨酸含量及心肌胶原含量较缺血组低,p<0.05,有统计学意义。免疫组化：1.肺充血组患儿Ⅰ、Ⅲ型胶原较缺血组减少。2.肺缺血组患儿心肌Ⅰ、Ⅲ型胶原表现为阳性染色。结论：1.两组先心于CPB术后不同时点MMP-3、-9先升高后降低,于术后3天降至低于术前的水平,但仍高于正常基础值。TIMP-1在肺充血组逐渐升高,于(?)。术后3天达最高,而肺缺血组在术后3天降低,两组TIMP-1均低于正常基础值。与cTnⅠ在术后显著升高,以后逐渐下降的趋势相符,说明CPB手术对心功能有一定的损害作用,但是术后心功能得到迅速恢复,并较术前明显改善,而MMPs/TIMPs的表达与cTnⅠ的浓度及心功能的变化存在着密切的相关关系。2.肺充血性先心心肌组织MMP-3、-9、TIMP-1 mRNA表达均高于肺缺血组,说明肺充血性先心的心功能降低较缺血类先心更为明显。3.肺充血性先心心肌胶原含量低于缺血组,可能与MMP-3、MMP-9活性增高有关。4.肺充血性先心心肌胶原Ⅰ、Ⅲ型染色均较缺血类先心弱,提示其胶原含量低于缺血类先心。说明由于MMPs升高,导致胶原降解增加,也可能与缺血类先心血氧降低有关。第三部分血管紧张素转换酶抑制剂对先天性心脏病围术期基质金属蛋白酶及抑制剂表达的影响及机制目的：于术前三天和术后应用ACEI治疗肺充血性先天性心脏病,并与仅常规治疗的肺充血性先心比较,了解术前和术后MMP-3、-9、TIMP-1的浓度及cTnⅠ的变化,并同时随访超声心动图,了解心功能的改变,为治疗充血性先心心力衰竭提供新的有效的治疗手段。方法：选取2009年1月～2009年8月在我院行体外循环手术治疗的室间隔缺损患儿共50例,分为两组,室间隔缺损常规治疗25例为对照组,室间隔缺损应用常规加ACEI治疗患儿25例为干预组。所有患儿于术前、术后一周行超声心动图检查,测LVEF、LVDs、LVDd、LVESV、LVEDV。干预组于术前三天给予依那普利(ACEI)0.2mg/kg/d,顿服,并于术后当天起继续应用依那普利(ACEI) 0.2mg/g/d,顿服。分别于术前、体外循环术后即刻、术后6h、24h、3d抽血测血浆MMP-3,-9、TIMP-1及cTnⅠ浓度及其变化。结果：超声心动图检查：1.术前两组之间LVEF、LVEDV、LVESV、LVDd、LVDs均无差异,p>0.05。2.术后两组之间LVEF、LVEDV、LVESV均无差异,但干预组LVDd、LVDs较对照组缩小,p<0.05,有统计学差异。3.术前与术后两组之间比较,除LVEF无变化外,LVEDV、LVESV、LVDd、LVDs均较术前减小,p<0.01,有显著统计学差异。实验室检测：ELISA:1.术前及术后各时点除干预组术后3天TIMP-1较高外,其余各参数干预组均低于对照组。术前、术后即刻两组cTnⅠ相比较无差异,但是术后6h、24h、3d,干预组cTnⅠ较对照组显著降低,p<0.01,有统计学意义。2.同一组内CPB前后不同时点比较：1)MMP-3：于CPB术后6-24小时达最高,以后逐渐下降,术后3天降至低于术前。2)MMP-9：于术后即刻显著升高,术后6小时达最高,以后逐渐下降,术后3天降至低于术前水平。3) TIMP-1:均表现为逐渐升高,于术后3天达到最高。4) cTnI:两组均于术后即刻明显升高,以后逐渐降低,并最终降至术前的水平,但是干预组较对照组降低的程度更为显著,p<0.05。结论：应用ACEI能有效调节MMPs/TIMPs的表达,术后MMPs的表达与cTnⅠ的降低相符,而TIMPs的表达则相反,说明通过MMPs/TIMPs的调节机制能进一步调节胶原代谢,抑制心室腔扩大,对于改善心功能具有一定的临床意义。更多还原

【Abstract】 Backgroud:Heart failure is a common complication in congenital heart disease during the peri-operation period. The most important thing for us is to diagnosticate early and treat timely. The majority mechanism of the development of heart failure is the active of neurohormonal mechanisms and cardiac remodeling. Cardiac remodeling includes the alteration in the myocardium cell and extracellular matrix. The alteration of the extracellular matrix is an important factor of cardiac remodeling. Matrix metalloproteinase are a family of zinc-dependent endopeptidases that degrade extracellular matrix proteins and make a major role in tissue remodeling. Matrix metalloproteinase and tissue inhibitor of metalloproteinase are specialized proteinases that regulate the metabolism of collegan. MMPs and TIMPs appear to be balanced in the normal myocardium thereby maintaining the integrity of the myocardium. In the patients of coronary artery diseases and hypertention, MMPs activity was increased but TIMPs decreased. It appears unbalanced between MMPs and TIMPs, break down the component and degradation of the ECM, develop the ventricular dilatation and cardiac dysfunction. The purpose of our study is to measure MMPs and TIMPs levels and expressions in plasma and cariac tissue during caridiopulmonary bypass in congenital heart diseases. To compare the expression of MMPs and TIMPs before and after the CPB operation between left-to-right shunt and right-to-left shunt of congenital heart diseases. To evaluate the effect of ACEI therapy in left-to-right shunt of congenital heart disease pre- and post-operation, measure the plasma MMPs and TIMPs levels before and after surgery. Part IThe Study on the Expression of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase in Congenital Heart DiseaseObjectivs:To measure the plasma MMP-3、-9 and TIMP-1 in congenital heart diseases. To detect the possible mechanism and compare the difference between left-to-right shunt and right-to-left shunt of congenital heart disease.Methods:From Jan.2009 to Aug.2009, there were 55 cases divided into three groups,10 cases of Pectus in the control group,25 cases of VSD in left-to-right group, and 20 cases of TOF in the right-to-left group. All the patients had ECG and Cardiac ECHO before surgery. We measure the LVEF, LVEDV, LVESV, LVDd, LVDs. The plasma levels of MMP-3,-9 and TIMP-1 were measured by radioimmunoassay (ELISA) before surgery.Results:ECHO:1. The LVEDV, LVESV, LVDd, and LVDs were larger in left-to-right shunt group than in control group.2. The LVEDV, LVESV, LVDd, and LVDs were smaller in right-to-left shunt group than in control group.3. In the group of left-to-right shunt, the LVEDV, LVESV, LVDd, and LVDs were larger than in right-to-left shunt group.Experimental:ELISA1. The plasma MMP-3 level was the highest in left-to-right group, but same between right-to-left group and control group.2. The plasma MMP-9 levels were higher both in left-to-right group and right-to-left group than control group. But it was the highest in the group of left-to-right group.3. The plasma TIMP-1 levels were lower both in left-to-right group and right-to-left group than control group. But it was the lowest in the group of right-to-left group.Conclusions:1. There might be different degree of heart failure in congenital heart diseases.2. Increased plasma MMP-3,-9 and decreased circulating TIMP-1 were determined in patients with congenital heart diseases.3. Increased plasma MMP-3,-9 and TIMP-1 were determined in group of left-to-righg shunt than in right-to-left group. It showed that the heart function was significantly lower in left-to-right group than in right-to-left group. PartⅡThe Study on the Effect of the Expression of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase during the Cariopulmonary Bypass in Congenital Heart DiseaseObjectivs:To measure the expression of MMP-3,-9 and TIMP-1 mRNA and the content of collagen in cardiac tissue of congenital heart diseases. To compare the plasma MMP-3,-9, TIMP-1 and cTnI levels during cardiopulmonary bypass in congenital heart diseases.Methods:From Jan.2009 to Aug.2009, there were 45 cases divided into two groups,25 cases of VSD in left-to-right group, and 20 cases of TOF in the right-to-left group. All the patients had ECG and Cardiac ECHO before and one week after surgery. We measured the LVEF, LVEDV, LVESV, LVDd, LVDs.The plasma levels of MMP-3,-9, TIMP-1 and cTnI were measured by ELISA before surgery and 6h,24h,3d after surgery respectively. The expression of MMP-3,-9 and TIMP-1 mRNA in cardiac tissue were detected by RT-PCR. We also measured the content of collegan in cardiac tissue.Results:ECHO:1. The LVEDV, LVESV, LVDd, and LVDs were significantly decreased after surgery in left-to-right group.2. The LVEDV, LVESV, LVDd, and LVDs were significantly increased after surgery in right-to-left group.3. There was no diference of LVEDV, LVESV, LVDd, and LVDs between left-to-right group and right-to-left group after surgery. Experimental:ELISA:1. The plasma MMP-3,-9 and TIMP-1 levels were significantly higher in left-to-right group than in right-to-left group before CPB and 6h,24h,3d after surgery. The level of cTnl was higher in left-to-right group before CPB, but lower after CPB.2. Compared at the different time after surgery in the same group:1) The plasma MMP-3,-9 levels were increased after CPB and extremely high at 6h after surgery. Then the level became lower and at the lowest level 3d after surgery.2) The plasma TIMP-1 level was increased after CPB and extremely high at the 3d after surgery in left-to-right group, but decreased at 3d after surgery in right-to-left group.3) The leverl of cTnⅠwas increased immediately after CPB and then decreased.RT-PCR:1. The expression of MMP-3、-9、TIMP-1 mRNA were significantly high in left-to-right group than in right-to-left group.2. The expression of MMP-3 mRNA was the lowest in left-to-right group. The expression of MMP-9 and TIMP-1 were extremely same.3. The expression of MMP-3 mRNA was the lowest in the right-to-left group, and TIMP-1 mRNA was the highest.Hydrolyzation Assay:The content of collegan in cardiac tissue was significantly low in left-to-right group.IHC:1. The content ofⅠ,Ⅲcollegan in cardiac tissue of left-to-right group were lower than in the right-to-left group.2. The content ofⅠ,Ⅲcollegan in cardiac tissue of right-to-left group were positive.Conclusions:1. The plasma MMP-3,-9 and cTnⅠlevels were increased after CPB and then decreased. The plasma TIMP-1 level was increased after CPB and extremely high at the 3d after surgery in left-to-right group, but decreased at 3d after surgery in right-to-left group. It showed that there might be cardiac injury because of CPB. But the heart function recovered rapidly after surgery. MMPs/TIMPs may play a role in regulating heart function as cTnⅠ.2. The expression of MMP-3、-9、TIMP-1 mRNA were significantly high in left-to-right group than in right-to-left group. It showed that marked heart dysfunction in left-to-right group.3. The content of collegan in cardiac tissue was significantly low in left-to-right group. It might because of the increased activation of MMP-3 and MMP-9.4. The content ofⅠ,Ⅲcollegan in cardiac tissue of left-to-right group were lower in left-to-right group than in right-to-left group. Part IIIThe Study on the ACEI Therapy of the Expression of Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinase during Cardiopulmonary Bypass in Congenital Heart DiseaseObjectivs:To evaluate the effect of ACEI therapy in left-to-right shunt congenital heart disease pre-, and post-CPB. To measure plasma MMP-3,-9, TIMP-1 and cTnⅠlevels during cardiopulmonary bypass between control group and ACEI group.Methods:From Jan.2009 to Aug.2009, there were 50 cases divided into two groups,25 cases of VSD in control group, and 25 cases of VSD with ACEI therapy in ACEI group. All the patients had ECG and Cardiac ECHO before and one week after surgery. We measure the LVEF, LVEDV, LVESV, LVDd, LVDs. The control group treated with standard therapy. The ACEI group with ACEI therapy used enalapril 0.2mg/kg/d by oral once daily three days before surgery. The group also used enalapril 0.2mg/kg/d by oral once daily from the day of surgery.The plasma levels of MMP-3,-9, TIMP-1 and cTnl were measured by ELISA before surgery and 6h,24h,3d after surgery respectively.Results:ECHO:1. There was no significantly difference of LVEDV, LVESV, LVDd, and LVDs between two groups before surgery.2. There was no difference of LVEF, LVEDV, LVESV between two groups after surgery. There were significant reduce of LVDd and LVDs in ACEI group.3. There were significant reduce of LVEDV, LVESV, LVDd and LVDs between two groups both before and after surgery. Experimental:ELISA:1. The plasma MMP-3,-9 and TIMP-1 levels were significantly lower in ACEI group than control group before CPB and 6h,24h,3d after surgery, expect for the TIMP-1 level at 3d after surgery. The level of cTnⅠwas no different between two groups before CPB and immediately after CPB, but decreased at 6h,24h and 3d. In ACEI group, the level of cTnⅠwas significantly lower than in control group.2. Compared at the different time after surgery in the same group:1) The plasma MMP-3,-9 levels were increased after CPB and extremely high at 6h after surgery in both groups. Then the level became lower and at the lowest level 3d after surgery.2) The plasma TIMP-1 level was increased after CPB and extremely high at 3d after surgery in both groups.3) The level of cTnⅠwas increased immediately after CPB and then decreased in two groups. Compared to the control group, the cTnⅠlevel of ACEI group was significantly lower.Conclusions:As a potential therapeutic, ACEI may convert the expression of MMPs and TIMPs. We got the same change of MMPs and cTnⅠbut diferent change of TIMPs and cTnⅠbetween two groups. It declared that the mechamism of MMPs/TIMPs can regulate the metabolism of collegan, depress the dilation of ventricle and improve the heart function.更多还原