干细胞调节心肌细胞代谢的分子显像研究

【作者】 蔡敏；

【导师】 何作祥；

【作者基本信息】 北京协和医学院 ， 影像医学与核医学， 2012， 博士

【摘要】 目的：骨髓间充质干细胞(bone marrow mesenchymal stem cells, BM-MSCs)是急性心肌梗死(acute myocardial infarction, AMI)干细胞移植治疗的理想供体之一,然而MSCs移植治疗缺血性心脏病的机制尚未完全阐明。我们提出假设,MSCs移植后心功能改善的原因不仅有MSCs的定向分化作用及血管生成作用,可能会有细胞能量底物代谢的增加,提高心肌细胞的能量来源,进而提高心肌收缩力。为此,本研究将细胞生物学与正电子发射计算机断层扫描-计算机断层扫描(PET-CT)单光子发射计算机断层扫描(SPECT)等分子影像检测手段相结合,并通过MRI检测,重点观察在体动物MSCs移植治疗AMI后心肌葡萄糖代谢、心肌血流灌注及心功能的改变,进而评价MSCs治疗缺血性心脏病的疗效及其机制。方法：24只中华小型猪(25±5kg)根据干预情况随机分为2组,干细胞移植组(MSCs组,n=12)及对照组(Control组,n=12)。通过开胸结扎左冠状动脉前降支30分钟建立AMI模型,体征平稳后于梗死周边心肌内注射自体骨髓MSCs(2×107,2ml),对照组以相同方法注射无血清的IMDM培养基。在1周及4周时行PET-CT及SPECT检测心肌葡萄糖代谢及心肌血流灌注情况,MRI检测心功能。术后4周为实验观察终点,组织取材后行H&E染色和Masson’s Trichrome染色观察梗死面积、炎症细胞浸润及心肌纤维化情况。结果：PET-CT评价心肌葡萄糖代谢,基线(1周)时MSCs组的最低FDG平均信号强度(MSI)低于Control组(22.10±3.18vs.35.70±3.02,P<0.05),总MSI亦低于Control组(1013.50±29.37vs.1084.00±21.15,P<0.05),其余各指标两组均无显著差异(P>0.05)。4周时,MSCs组左室心肌18F-FDG最低MSI较1周时有明显提高(34.00±4.25vs.22.10±3.18,P<0.01),总MSI较1周时亦明显提高(1075.50±28.30vs.1013.50±29.37,P<0.01),而SRS(20.20±2.24vs.23.80±1.58,P<0.05)及SRS%(29.80±3.31vs.35.10±2.34,P<0.05)较1周时均有减低；左心室梗死区(MSI低于70%的范围内)节段的总MSI及各节段平均MSI较1周时有显著增加(384.60±37.13vs.323.60±18.99,P<0.01；56.25±3.54vs.48.14±2.71,P<0.01);MSCs组左心室代谢缺损面积(Defect, cm2)、缺损范围(Extent,%)及总灌注缺损范围(TPD,%)在4周时虽较1周时有所减低,但无显著性差异(27.20±4.06vs.28.90±2.48；37.70±3.79vs.42.40±2.46；31.70±3.52vs.36.00±2.57；P值均>0.05)。在Control组中,以上参数4周时均较1周时有改善,但均无统计学差异(P>0.05)。MSCs组最低MSI及左心室总MSI在4周与1周时的增加量与Control组相比有显著差异(11.90±2.93vs.1.70±2.00,P<0.05；85.80±19.50vs.1.10±17.09,P<0.01)。MSCs组及Control组分别有6例及5例动物接受SPECT心肌血流灌注检查,1周时MSCs组与Control组相比,各项参数无显著差异(P>0.05)；分别在1周及4周时对两组进行自身配对检验,各项心肌血流灌注指标及灌注缺损面积均无显著改变(P值均>0.05)。MRI评价心功能,1周时两组心功能参数无显著差异(P>0.05)；MSCs组在4周时,左室射血分数(LVEF)较1周时有显著增加(54.41±2.62vs.47.54±2.43,P<0.01),左室收缩末期容积(ESV)有显著减低(22.85±1.91vs.27.07±1.67,P<0.01),左心室每搏输出量(SV)及心输出量(CO)均较1周时均有显著增加(29.35±1.84vs.26.52±1.46,P<0.05；2.23±0.14vs.1.96±0.13,P<0.05)：Control组1周及4周时以各项心功能标均无统计学差异(P>0.05)。结论：经心肌内注射骨髓MSCs治疗AMI,4周后心功能明显改善,心肌葡萄糖代谢显著提高,而心肌血流灌注未见明显改善。推测心功能的改善与心肌糖代谢增加相关。目的：干细胞移植治疗缺血性心脏病的机制尚未完全阐明,本课题通过检测猪急性心肌梗死(acute myocardial infarction, AMI)后骨髓间充质干细胞(mesenchymal stem cells, MSCs)移植对左心室心肌整体及局部葡萄糖代谢、心功能的影响,并通过分子生物学手段对葡萄糖转运体、葡萄糖代谢相关酶及可能参与MSCs旁分泌作用的信号转导通路进行初步的研究,以探索MSCs移植治疗AMI的疗效及机制。方法：32只中华小型猪按干预情况及观察终点随机分为3组：MSCs-1周组(n=8)、MSCs-4周组(n=12)及Control-4周组(n=12)。分离并培养猪MSCs,3-4周后开胸结扎左冠状动脉前降支30分钟建立AMI模型,体征平稳后,MSCs组(MSCs-1周组,MSCs-4周组)于梗死周边心肌内注射自体骨髓MSCs (2×107,2ml),对照组以相同方法注射无血清的IMDM培养基。分别在1周及4周时行PET-CT检测心肌葡萄糖代谢情况,并行MRI检测心功能。按不同的观察终点处死动物并取材,通过实时定量聚合酶链反应(real time-PCR)检测MSCs注射区域葡萄糖转运蛋白(GLUT1、GLUT4)、葡萄糖代谢相关酶(PFK、GAPDH)及mTOR信号通路相关基因表达的水平。结果：PET-CT检测示,基线(1周)时MSCs-1周组与MSCs-4周组各指标无显著差异,而MSCs-4周组的最低MSI低于Control-4周组(22.10±3.18vs.35.70±3.02,P<0.05),总MSI亦低于Control-4周组(1013.50±29.37vs.1084.00±21.15,P<0.05)。MSCs-4周组4周时左室心肌18F-FDG摄取的最低MSI较1周时有明显提高(34.00±4.25vs.22.10±3.18,P<0.01),总MSI较1周时亦明显提高(1075.50±28.30vs.1013.50±29.37,P<0.01),SRS(20.20±2.24vs.23.80±1.58,P<0.05)及SRS%(29.80±3.31vs.35.10±2.34,P<0.05)较1周时均有减低；左心室梗死区内总MSI及平均MSI较1周时有显著增加(384.60±37.13vs.323.60±18.99,P<0.01；56.25±3.54vs.48.14±2.71,P<0.01)；在Control-4周组中,以上参数在4周时与1周时比较,其改变无统计学意义(P>0.05)。局部左心室代谢评价：MSCs-4周组在4周时,以下节段MSI较1周时有显著增加：左心室前壁近心尖段(32.00±5.35vs.44.10±5.90,P<0.05)、前壁中段(57.40±4.00vs.65.30±4.66,P<0.05)、间隔近心尖部(52.00±2.55vs.61.60±2.67,P<0.05)、前间隔中段(62.80±2.85vs.69.50±2.17,P<0.05),其余节段MSI均未见显著性增加；而Control-4周组中,各节段MSI在1周及4周时均无明显差异。MRI评价心功能,基线时3组心功能参数无显著差异(P>0.05)。MSCs组在4周时,左室射血分数(LVEF)较1周时有显著增加(54.41±2.62vs.47.54±2.43,P<0.01),左室收缩末期容积(ESV)有显著减低(22.85±1.91vs.27.07±1.67,P<0.01),左心室每搏输出量(SV)及心输出量(CO)均较1周时均有显著增加(29.35±1.84vs.26.52±1.46,P<0.05；2.23±0.14vs.1.96±0.13,P<0.05);Control组1周及4周时以各项心功能标均无统计学差异(P>0.05)。Real-time PCR检测显示,MSCs-4周组葡萄糖转运蛋白(GLUT1、 GLUT4)、葡萄糖代谢相关酶(PFK、GAPDH)及mTOR信号通路下游蛋白p70s6k的基因表达与Control-4周组及MSCs-1周组相比,均显著增加(P值均<0.05)结论：AMI后心肌内注射MSCs可提高心肌细胞葡萄糖代谢及心功能,注射MSCs的区域心肌细胞葡萄糖转运体及糖酵解相关酶表达上调,mTOR通路下游分子p70s6k表达显著升高,提示MSCs可能通过旁分泌作用激活该通路,促进心肌葡萄糖代谢及细胞生长,进而增加心肌收缩、促进心功能改善。目的：门控单光子发射计算机断层(SPECT)心肌灌注显像不仅可以评价心肌血流灌注情况,亦可评价心功能,测定心室腔容积,对心衰患者的预后评价具有重要意义。本研究旨在探讨静息门控SPECT心肌灌注显像和其他临床变量如体重指数对慢性心力衰竭(CHF)患者心脏性死亡的预测价值,筛选心脏性死亡的预测因子。方法：73例确诊为慢性心力衰竭(CHF)且左室射血分数(LVEF)<40%的住院患者(年龄50.7±16.5岁；男60例,女13例；缺血性心衰患者25例,非缺血性心衰患者48例),行99mTc-MIBI静息门控SPECT心肌灌注显像,以心脏性死亡作为心脏事件随访所有患者。用Cox比例风险回归分析法检测心脏性死亡的预测因子并用Kaplan-Meier方法估计生存概率。结果：随访时间为18.6±8.5月(1.1～30.0月)。随访期间内共14例患者(19.2%)发生心脏性死亡。单因素Cox回归分析示体重指数(BMI, Wald4.66, P<0.05)、脑钠肽(BNP, Wald4.87, P<0.05)门控LVEF (Wald4.64, P<0.05),静息运动总评分(SMS, Wald4.43, P<0.05),静息灌注总评分(SRS, Wald4.56, P<0.05)以及灌注异常范围(Def Ext, Wald4.48, P<0.05)为心脏性死亡预测因素。将以上因素纳入多因素Cox回归分析,结果显示BMI (23.3±4.1kg/m2, Wald5.02, P<0.05,RR=0.85)及SRS (11.8±11.5, Wald5.33, P<0.05, RR=1.05)为独立心脏性死亡预测因素。BMI的最佳阈值为25Kg/m2, BMI<25Kg/m2的CHF患者(46例)及BMI≥25Kg/m2者(27例)预后有显著差异(P<0.05),BMI<25Kg/m2者死亡率高(28.3%vs.3.7%,P<0.05)且累计生存率明显高于BMI≥25Kg/m2者(Log-rank6.11, P<0.05); ROC曲线测得SRS最佳阈值为11,SRS≤11的CHF患者(43例)与SRS>11者(30例)两组间死亡率无明显差异(P=0.051),而两组累计生存率有显著差异,SRS≤S11者高于SRS>11者(Log-rank6.83, P<0.01)。 BMI<25Kg/m2同时SRS>11的CHF患者累积生存率明显减降低(Log-rank18.50, P<0.001)。结论：静息门控SPECT心肌灌注显像对CHF患者有预后价值。BMI<25Kg/m2及SRS>11为发生心脏性死亡的独立危险因素,BMI<25Kg/m2且SRS>11的患者发生心脏性死亡的风险更高,应加强治疗及护理。更多还原

【Abstract】 Obiective:Bone morrow mesenchymal stem cells (BM-MSCs) have been one of the optimal candidate cells for acute myocardial infarction (AMI) in recent years, however, the mechanism of MSCs transplantation in curing ischemic heart disease has not yet been fully. We proposed, MSCs transplantation improves cardiac function not only by differentiation and angiogenesis of MSCs, but also improve the metabolism of cardiocytes energy substrate, and then improve myocardial contraction. In this study, we will in vivo observe the changes of myocardial glucose metabolism, myocardial perfusion and cardiac function after MSCs transplantation in swine with AMI by means of cell biology and molecular imaging methods including positron emission tomography-computer tomography (PET-CT), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI), in the purpose of evaluating the effect and mechanism of MSCs transplantation treating ischemic heart disease.Method:Twenty-four Chinese mini-swine were randomized into2groups of MSCs transplantation group (MSCs group; n=12) and Control group (n=12). Myocardial infarction was induced in swine hearts by occlusion of the left anterior descending artery (LAD). Thirty minutes later, the MSCs group received autologous BM-MSCs transplantation through intramyocardial injection into the peri-infarcted areas and Control group were subjected to cell culture medium in the same way. After1week and4weeks, myocardial glucose metabolism, myocardial perfusion and cardiac function were evaluated in the two groups through PET-CT, SPECT and MRI. On the end point (the4th week), H&E stain and Masson’s Trichrome stain were performed to observe the extent of infarction area, inflammatory cells infiltration and myocardial fibrosis.Results:As evaluated by PET, on the baseline, the minimum FDG mean signal intensity (MSI) in MSCs group was below Control group (22.10±3.18vs.35.70±3.02, P<0.05), and the summed MSI was below Control group (1013.50±29.37vs.1084.00±21.15, P <0.05). Compared to the1st week, the minimum MSI in MSCs group was increased obviously (34.00±4.25vs.22.10±3.18, P<0.01) on the4th week, and also the summed MSI (1075.50±28.30vs.1013.50±29.37, P<0.01); summed rest score (SRS) and SRS%were decreased on the4th week compared to the1st week (20.20±2.24vs.23.80±1.58, P<0.05;29.80±3.31vs.35.10±2.34, P<0.05); the summed MSI in left ventricular infarction area (in area MSI below70) and average MSI were also increased (384.60±37.13vs.323.60±18.99, P<0.01;56.25±3.54vs.48.14±2.71, P<0.01); the metabolism defect area, defect extent and TPD the4th week were lower than those was on the1st week, but there were no significant differences (27.20±4.06vs.28.90±2.48;37.70±3.79vs.42.40±2.46;31.70±3.52vs.36.00±2.57; P>0.05). However, in the Control group, the variables mentioned above had no statistics differences in the endpoint than in the baseline (all P<0.05). The differences of minimum MSI between1st week and4th week in the MSCs group significantly high than the Control group (11.90±2.93vs.1.70±2.00, P<0.05), and also the differences of summed MSI between the two groups (85.80±19.50vs.1.10±17.09, P<0.01). Only6swine in the MSCs group and5swine in the Control group evaluated myocardial perfusion by SPECT. On the baseline, the perfusion variables had no significant differences between the two groups (P>0.05), and there were no differences in variables reflecting myocardial perfusion and defect area between baseline and endpoint in the two groups (P>0.05). As evaluated by MRI, the cardiac functional parameters had no significant differences in the two groups on the baseline. In the MSCs groups, left ventricular ejection fraction (LVEF) was increased significantly (54.41±2.62vs.47.54±2.43, P<0.01) and end-systolic volume (ESV) was significantly reduced (22.85±1.91vs.27.07±1.67, P<0.01) on the4th week compared to the1st week; stroke volume (SV) and cardiac output (CO) in the4th week also increased significantly (29.35±1.84vs.26.52±1.46, P<0.05;2.23±0.14vs.1.96±0.13, P<0.05). In the Control group, there were no significant differences in the cardiac functional parameters on the baseline and endpoint(P>0.05).Conclusion:Four weeks after MSCs transplantation for curing AMI, cardiac function and myocardial glucose metabolism improved significantly; however, obvious myocardial perfusion improvement was not seen. It is speculated that the cardiac improvement is associated with the enhancement of myocardial glucose metabolism. Objective:The mechanism of stem cells transplantation in curing ischemic heart diseases has not been clarified. The aim of this study is to investigate the effect and mechanism of bone marrow mesenchymal stem cells (BM-MSCs) transplantation in acute myocardial infarction (AMI) by means of detecting glucose metabolism in global left ventricular myocardium and regional myocardium, combined with assessment of cardiac function, and also to study the changes of glucose transporters, glucose metabolism-related enzymes and the signal transduction pathway which may participate in the MSCs paracrine process.Methods:Thirty-two Chinese mini-swine were randomized into3groups of MSCs transplantation1-week group (MSCs-lw group; n=8), MSCs transplantation4-week group (MSCs-4w group; n=12) and Control group (n=12). BM-MSCs were separated and cultured for3-4weeks and then myocardial infarction was induced in swine hearts by occlusion of the left anterior descending artery (LAD). Thirty minutes later, the MSCs groups (MSCs-lw group and MSCs-4w group) received autologous MSCs (2×107,2ml) transplantation through intramyocardial injection into the peri-infarcted areas and Control group were subjected to cell culture medium in the same way. Positron emission tomography-computer tomography (PET-CT) and magnetic resonance imaging (MRI) were performed on the1st week and4th week. The swine were killed on the endpoints (1st week or4th week), and the gene expression of glucose transporters (GLUT1, GLUT4), glucose metabolism-related enzymes (PFK, GAPDH) and the proteins in mTOR signal transduction pathway in MSCs injection area were measured by real-time polymerase chain reaction (PCR).Results:As shown by PET-CT, all the variables had no differences in the MSCs-lw group and MSCs-4w group on the baseline. The minimum FDG mean signal intensity (MSI) in MSCs-4w group was below Control-4w group (22.10±3.18vs.35.70±3.02, P <0.05), and the summed MSI was below Control-4w group (1013.50±29.37vs.1084.00±21.15, P<0.05). Compared to the1st week, the minimum MSI in MSCs-4w group was increased obviously (34.00±4.25vs.22.10±3.18, P<0.01) on the4th week, and also the summed MSI (1075.50±28.30vs.1013.50±29.37, P<0.01); summed rest score (SRS) and SRS%were decreased on the4th week compared to the1st week (20.20±2.24vs.23.80±1.58, P<0.05;29.80±3.31vs.35.10±2.34, P<0.05); the summed MSI in left ventricular infarction area (in area MSI below70) and average MSI were also increased (384.60±37.13vs.323.60±18.99, P<0.01;56.25±3.54vs.48.14±2.71, P <0.01). However, in the Control-4w group, the variables mentioned above had no statistics differences in the endpoint than in the baseline (all P<0.05). Metabolic evaluation in regional left ventricular showed that MSI increased in these segments on the4th week compared to the1st week:apical-anterior segment (32.00±5.35vs.44.10±5.90, P<0.05), mid-anterior segment (57.40±4.00vs.65.30±4.66, P<0.05), apical-septal segment (52.00±2.55vs.61.60±2.67, P<0.05) and mid-anteroseptal segment (62.80±2.85vs.69.50±2.17, P<0.05), and MSI in other segments didn’t increase significantly (P>0.05). However, there were no differences in MSI between the1st week and4th week in the Control-4w group. As evaluated by MRI, the cardiac functional parameters had no significant differences in the three groups on the baseline (P>0.05). In the MSCs-4w group, left ventricular ejection fraction (LVEF) was increased significantly (54.41±2.62vs.47.54±2.43, P<0.01) and end-systolic volume (ESV) was significantly reduced (22.85±1.91vs.27.07±1.67, P<0.01) on the4th week compared to the1st week; stroke volume (SV) and cardiac output (CO) in the4th week also increased significantly (29.35±1.84vs.26.52±1.46, P<0.05;2.23±0.14vs.1.96±0.13, P<0.05). In the Control-4w group, there were no significant differences in the cardiac functional parameters on the baseline and endpoint (P>0.05). In the MSCs-4w group, real-time PCR analysis showed positive up-regulation of GLUT1, GLUT4, PFK, GAPDH and p70s6k (a downstream protein in mTOR signal transduction pathway) compared to the Control-4w group and the MSCs-lw group (all P<0.05).Contusion:Intramyocardial injection of MSCs after AMI could improve cardiocytes glucose metabolism and improve cardiac function. The gene expression of glucose transporters (GLUT1, GLUT4), glucose metabolism-related enzymes (PFK, GAPDH) and p70s6k in MSCs injection area are up-regulated on the4th week after MSCs transplantation. It indicates that MSCs may active mTOR signal transduction pathway through paracrine effect to promote myocardial glucose metabolism and cardiocytes growth and then give rise to improved myocardial contraction and enhanced cardiac function. Objective:Gated single photon emission computed tomography (SPECT) myocardial perfusion imaging has proven to be invaluable not only in assessing myocardial perfusion, but also in providing functional and volumetric information. The aim was to investigate the value of rest gated-SPECT myocardial perfusion imaging for predicting cardiac death in patients with chronic heart failure (CHF).Methods:Seventy-three consecutive hospitalized patients (50.7±16.5years,60men) with defined diagnosis of CHF (25ischemic CHF and48non-ischemic CHF) and echocardiography left ventricular ejection fraction (LVEF)<40%, who underwent rest99mTc-MIBI gated SPECT myocardial perfusion imaging, were followed up for18.6±8.5months (range,1.1-30.0months). Only cardiac death during follow-up served as the endpoint. Cox proportional hazard regression analysis was applied to determine independent predictors of cardiac death and Kaplan-Meier method was applied to estimate the probability of survival with CHF.Results:During the follow-up period,14(19.2%) cardiac deaths occurred in the73patients. Univariate Cox analysis showed that body mass index (BMI,23.3±4.1Kg/m2, P<0.05), brain natriuretic peptide (BNP, P<0.05), gated-LVEF (20.8%±7.9%, P <0.05), summed motion score (P<0.05), summed rest score (SRS, P<0.05) and defect extent (P<0.05) were significant predictors. When the above predictors were applied to multivariate Cox analysis, BMI (P<0.05, Hazard Ratio=0.85) and SRS (P<0.05, Hazard Ratio=1.05) showed predictive values for future cardiac death. The optimal threshold of BMI was25Kg/m2, the difference of cumulative survival between patients with BMI<25Kg/m2and those with BMI≥25Kg/m2was significant (P<0.05) and patients with BMI<25Kg/m2had lower survival. The optimal threshold of SRS was set as11, the difference of cumulative survival between patients with SRS≤1and those with SRS>11was significant (P<0.01), and patients with SRS>11had lower survival. The patients with BMI<25Kg/m and SRS>11simultaneously had the lowest survival in the73patients with CHF (P<0.001).Conclusions:The present study indicates that the rest gated SPECT myocardial perfusion imaging gives prognostic information in patients with CHF. Both BMI and SRS are predictors for future occurrence of cardiac death, and patients with BMI<25Kg/m2and SRS>11simultaneously should be treated and cared for intensively.更多还原