基质弹性在确定急性心肌梗死后骨髓单个核细胞移植最佳时机中的作用
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摘要
前言
骨髓源细胞移植治疗急性心肌梗死(AMI)的最佳时机问题已引起学术界的广泛关注。已有少量研究表明,移植时机的选择是影响移植疗效的重要因素。目前学术界倾向于最佳移植时机可能出现在AMI后一周左右的观点。然而,其潜在机制尚不明确。
近来越来越多的证据显示,干细胞周围的物理微环境(主要是基质的弹性或硬度,用弹性模量[Elastic Modulus,E]描述)决定着其生物学特性和转归,即由不同弹性基质诱导分化而来的细胞在形态和功能上更接近于宿主组织的细胞。具体来说,类似于脑组织的软基质能够诱导骨髓源干细胞向神经元样细胞分化;类似于肌肉的中等弹性基质能够诱导其发生肌原性分化;类似于骨胶原的硬基质能够刺激其骨原性分化。
同时,考虑到心肌梗死后由大量心肌坏死和细胞外基质的沉积诱发的心肌重构导致梗死心肌发生由软到硬的时间依赖性弹性变化的科学事实,据此,我们提出了AMI后一定时间窗内的心肌弹性可能更有利于移植细胞向有利于心功能改善的方向分化的科学假说,这一时段即为最佳移植时机。
本研究旨在通过一系列研究验证该假说及其前提的正确性,对于这一科学命题的论述将有益于骨髓源细胞移植治疗AMI的时机选择及最佳移植时机潜在机制的阐明。
第一部分移植时间对急性心肌梗死后骨髓单个核细胞移植疗效和安全性影响的荟萃分析
目的通过Meta分析的亚组分析系统评价移植时间对急性心肌梗死(AMI)后骨髓单个核细胞(BMMNCs)移植疗效和安全性的影响。
方法系统检索PubMed、Medline、Cochrane EBM Reviews、EMBASE、BIOSIS等数据库。以AMI症状出现24小时内成功实施经皮冠状动脉介入治疗(PCI)者为研究对象;试验设计体现随机对照原则;移植细胞种类为非动员的自体BMSCs;具有3个月以上的随访资料。样本总量小于10的研究未被纳入。
结果共检出相关文献349篇,筛选出符合纳入标准的文献共9篇,涉及7项研究,总病例数660例。其中心肌梗死后24小时内移植亚组271例,4-7天移植亚组389例,与对照组相比,BMMNCs移植可显著改善左室射血分数(LVEF)和左室收缩末容积(LVESV)。亚组综合效应分析显示,4-7天移植亚组能够明显提高LVEF和减少LVESV(LVEF:权重均差[WMD]=4.63%,95%CI 1.00%-8.26%,p=0.01;LVESV:标化均差[SMD]=—0.28,95%CI—0.53-—0.02,p=0.03),而24小时内移植亚组仅显示出有益趋势,两亚组相比,4-7天移植亚组较24小时内移植亚组可能更有利于LVEF和LVESV的改善,但两亚组间统计学差异无显著性(p＞0.05)。而移植时间对左室舒张末容积(LVEDV)呈中性影响。死亡、支架内或罪犯血管再狭窄、再次心肌梗死、再次血运重建、因心力衰竭再入院等独立不良事件及死亡、再次血运重建、再次心肌梗死或因心力衰竭再入院的联合终点发生率均显示出BMMNCs移植治疗的有益趋势(比值比[OR]均＜1,但p均＞0.05)。与对照组相比,4-7天移植亚组能够明显降低再次血运重建率(OR=0.60,95%CI 0.37-0.97,p=0.04)、死亡或再次心肌梗死的联合发生率(OR=0.32,95%CI 0.11-0.95,p=0.04)及死亡、罪犯血管再狭窄、再次心肌梗死或室性心律失常的联合发生率(OR=0.59,95%CI 0.36-0.96,p=0.03),而这些指标在24小时内移植亚组与对照组间无显著差异;两亚组间相比,4-7天移植亚组的再次血运重建率明显低于24小时内移植亚组(交互p=0.02)。
结论自体BMMNCs移植治疗AMI安全可行,移植时间是移植疗效和安全性的重要影响因素,AMI后4-7天进行细胞移植较24小时内移植其疗效和安全性可能更佳。
第二部分心肌梗死后不同时期心肌弹性、血清VEGF浓度及心脏功能变化
目的探讨小鼠心肌梗死后心肌弹性、血清VEGF浓度和心脏功能的时间变化规律及其潜在联系,为进一步分析心肌弹性和VEGF在骨髓单个核细胞(BMMNCs)分化中的作用和相互作用创造条件。
方法六周龄雄性BALB/c小鼠48只,体重20-25g,采用结扎左冠状动脉制成心肌梗死模型,以假手术动物为对照。分别于心肌梗死后1小时、24小时、7天、14天和28天,行心脏功能和左心室腔内压力检测后,采用ELISA方法检测血清VEGF浓度。每组送检3个标本,应用原子力显微镜检测梗死心肌弹性模量(E);其余心脏组织制成石蜡切片行苏木素-伊红(HE)染色和Mallary纤维染色。多组间两两比较采用单因素方差分析,p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件包进行数理分析。
结果梗死后1小时心肌弹性模量轻微降低(由对照组心肌的17.94 kPa降至16.60 kPa,p＞0.05);24小时后降至最低值(4.21 kPa,p＜0.001),其后梗死心肌弹性逐渐增加,由梗死后7天的31.38 kPa增至4周后的90.22 kPa(与对照组相比,p均＜0.001)。梗死后不同时间点血清VEGF检测显示,心肌梗死后1小时VEGF浓度由正常对照的38.58 pg/ml升至49.44 pg/ml(p=0.024),至24小时达峰值(96.30 pg/ml,p=0.005),心肌梗死后7天、14天和28天逐渐下降(VEGF浓度分别为50.56 pg/ml[p=0.014]、43.89 pg/ml[p＞0.10]、39.82pg/ml[p＞0.10],p值均为与对照组相比)。组织病理染色显示,心肌梗死后24小时,出现大量心肌细胞坏死,炎性细胞浸润与各组相比最为明显;至梗死后7天,胶原纤维逐渐增多,早期疤痕组织开始形成;梗死后14天和28天,纤维疤痕组织逐渐成熟。心肌梗死后心脏功能和左心室腔内压力的改变表现出与上述理化参数相似的变化趋势,AMI后24小时LVEF、FS和LVESP降至最低点(VEGF,0.50对0.86[对照组],p＜0.01;FS,0.25对0.55,p＜0.01;LVESP,99.30 mmHg对144.67 mmHg,p＜0.01),且此时LVEDP亦达到首个峰值(12.35 mmHg对3.89 mmHg[对照组],p＜0.01)。
结论小鼠心肌梗死后心肌弹性模量、血清VEGF浓度和心脏功能均表现出时间依赖性相关变化且相互之间保持较高的一致性。梗死心肌的弹性模量经历了先降低后逐渐增高的变化过程,AMI后24小时心肌弹性模量降至最低,而此时血清VEGF浓度达到峰值;心肌梗死区域炎症反应亦最为明显,且心肌梗死后心脏功能受抑最为明显。
第三部分基质弹性和VEGF在骨髓单个核细胞向内皮祖细胞分化中的作用
目的应用可以模拟梗死后不同时期心肌弹性的弹性可控性细胞培养体系,探讨基质弹性及VEGF在诱导骨髓单个核细胞(BMMNCs)向内皮祖细胞(EPC)分化中的作用和相互作用。
方法采用不同配比浓度的丙烯酰胺和甲叉双丙烯酰胺制成不同硬度的聚丙烯酰胺凝胶;利用原子力显微镜筛选出能模拟心肌梗死后1小时、24小时、7天、14天和28天心肌弹性模量的凝胶用于细胞培养。采用密度梯度离心法分离BMMNCs,每一弹性模量的基质胶上分别采用0ng/ml、2.5ng/ml、10ng/ml、20ng/ml等四个浓度的VEGF进行细胞培养。7天后进行乙酰化低密度脂蛋白(ac-LDL)吞噬/荆豆凝集素-1(UEA-1)结合双阳性细胞免疫荧光检测及EPC表面标志物CD133、CD45、VEGFR2流式细胞分析。同一基质弹性或同一浓度VEGF下的数据分析采用单因素方差分析,基质弹性和VEGF对促细胞分化的相互作用分析应用两因素方差分析,p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件进行数理统计。
结果筛查出4种不同浓度凝胶的弹性模量(分别约为4kPa、15kPa、42 kPa和72kPa)可分别模拟梗死后24小时、1小时、7-14天和14-28天的梗死心肌弹性。FITC-UEA-1/DiI-AcLDL双阳性细胞免疫荧光检测显示,在较高浓度VEGF(10-20ng/ml)培养条件下,贴壁细胞双阳性表达率在各弹性基质间无显著差异(p均＞0.05);当VEGF浓度降至2.5 ng/ml,42kPa基质胶的双阳性表达率显著高于15kPa(72.44%对52.44%,p=0.04),且双阳性细胞数量远大于其他弹性凝胶(p＜0.01);在无VEGF的培养条件下,42kPa在双阳性细胞表达率和细胞数量上表现出较其他弹性更大的优势,此外,4kPa和15kPa双阳性细胞表达率较72kPa显著降低(p均＜0.05)。两因素方差分析显示,基质弹性和VEGF浓度对BMMNCs向EPC的分化存在显著交互作用(p＜0.01),分别控制VEGF浓度和基质弹性的影响后,42kPa的基质弹性和较低浓度的VEGF(2.5-10 ng/ml)较其他弹性和浓度具有更高的的双阳性细胞表达率。此外,流式细胞分析表明,低浓度VEGF(2.5ng/ml)条件下,EPC特征性表面标记物CD45(-)/VEGFR-2(+)/CD133(+)细胞表达率以仍以42 kPa为最高(1.94%)。
结论基质弹性在促进BMMNCs向EPC分化中发挥着重要作用,42 kPa(模拟心肌梗死后7-14天心肌弹性)的促细胞内皮系分化效应优于其他弹性模量。基质弹性与VEGF浓度在促进BMMNCs向EPC分化过程中可能发挥着协同作用。
第四部分心肌梗死后骨髓单个核细胞最佳移植时机及其潜在机制
目的通过对心肌梗死不同时期BMMNCs移植后心脏功能改善状况的研究,探讨心肌梗死后BMMNCs移植的最佳时机及其潜在机制。
方法采用密度梯度离心法分离BALB/c小鼠BMMNCs。分别于心肌梗死后1小时、24小时、7天、14天和28天实施梗死区域BMMNCs局部注射,以相应时间点M199注射为对照,各组于心肌梗死建模后2月,应用小动物超声成像系统和心腔导管压力测试系统对实验动物实施左心功能和左心室腔内压力测定,通过vWF免疫组化方法检测移植区域毛细血管密度。移植组与各对照组比较用成组t检验,多组间两两比较应用单因素方差分析(ANOVA),p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件进行数理统计。
结果与对照组相比,心肌梗死后24小时至14天实施BMMNCs移植可显著提高梗死区域毛细血管密度(对照组:1.32个/高倍镜[HP];AMI后24小时移植组:2.60个/HP;7天移植组:4.60个/HP;14天移植组:3.80个/HP;p均＜0.001),其中以7天移植组的毛细血管密度为最高(两者相比p=0.124)。各移植组LVEF、FS、LVEDP、LVESP及±dp/dt的绝对增量相比,亦显示出相似变化,且以AMI后7天和14天移植组改善最为明显,优于24小时移植(ΔLVEF:20.95%对11.41%,p=0.025;ΔFS:13.47%对7.13%,p=0.023;ΔLVEDP:-15.94 mmHg对-8.17 mmHg,p＜0.05;+Δdp/dt:9001.02mmHg/s对4891.53 mmHg/s,p＜0.05),而与14天移植组相比,统计学差异无显著意义(ΔLVEF:18.82%,p=0.57;ΔFS:11.63%,p=0.46;ΔLVEDP:-15.34 mmHg;ΔLVESP:9.19 mmHg;-Δdp/dt:-4650.71 mmHg/s;+Δdp/dt:8265.35 mmHg/s,p均＜0.05)。ΔLVEF、ΔFS、ΔLVEDP及ΔLVESP等心脏功能相关参数和毛细血管密度间存在高度线性相关(相关系数均＞0.75,p均＜0.01)。此外,7天移植组和14天移植在LVIDd和LVIDs的改善方面呈现的有利趋势亦最为明显,但与其他各组相比差异显著性无统计学意义(p均＞0.05)。
结论AMI后BMMNCs移植治疗的最佳时机出现在心肌梗死后7-14天。在这一时段,细胞移植可最大限度的改善心脏功能和左心室腔内压力,这可能与此时最为适宜的物理微环境(梗死心肌弹性)促进移植区域毛细血管密度的增加进而显著改善了梗死心肌的顺应性有关。
PREFACE
Determining which time point is optimal for bone marrow-derived cells transplantation for acute myocardial infarction(AMI) has attracted a great deal of attention.Studies have verified the interaction between cell treatment effect and transfer timing and have suggested that the optimal time frame for bone marrow-derived cells therapy might be about one week after AMI.
However,the potential mechanism underlying the time-dependent therapeutic response remains unclear.Recently,a growing body of in vitro evidence has suggested that stem cells are able to feel and respond to the stiffness of their microenvironment to commit to a relevant lineage,indicating that soft matrices that mimic brain are neurogenic,stiffer matrices that mimic muscle are myogenic and comparatively rigid matrices that mimic collagenous bone prove osteogenic.
Simultaneously,considering the fact that the myocardium post-infarction experiences a time-dependent stiffness change from flexible to rigid as a result of myocardial remodelling following tissue necrosis and massive extracellular matrix deposition,we presume that the myocardial stiffness within a certain time frame (possibly one week post-AMI) after infarction might provide a more favourable physical microenvironment for the phenotypic plasticity and functional specification of engrafted bone marrow-derived cells committed to some cell lineages,such as endothelial cells,vascular smooth muscle cells or cardiomyocytes.The beneficial effect facilitates angiogenesis and myocardiogenesis in the infarcted heart,and subsequently leads to more amelioration of cardiac functions.
The present study aimed to verify the scientific hypothesis.If the hypothesis were true,it would be of great help to understand the mechanism underlying the optimal timing for bone marrow-derived cells transplantation and to establish a direction for the time selection of cell therapy.
PART ONE Impact of Timing on Efficacy and Safety of Bone Marrow Mononuclear Cells Transplantation in Acute Myocardial Infarction: A Pooled Subgroup Analysis of Randomised Controlled Trials
Objectives
To investigate the impact of timing on efficacy and safety of bone marrow mononuclear cells(BMMNCs) transfer in patients with acute myocardial infarction (AMI) by a pooled subgroup analysis of randomised controlled trials.
Methods
A systematic literature search of PubMed,MEDLINE and Cochrane EBM databases was made on randomised controlled trials with at least 3-month follow-up data for patients with AMI undergoing emergent percutaneous coronary intervention(PCI) and receiving BMMNCs transfer thereafter.
Results
A total of 7 trials with 660 patients were available for analysis.Compared to baseline level,BMMNCs transfer at day 4 to 7 post AMI significantly improved left ventricular ejection fraction(LVEF)(4.63%increase,95%confidence interval[CI] 1.00%to 8.26%,p=0.01),reduced left ventricular end-systolic volumes(LVESV) (95%CI—0.53 to—0.02,p=0.03),and decreased the incidences of revascularization (odd ratio[OR]=0.60,95%CI 0.37 to 0.97,p=0.04) and the cumulative clinical events of death or recurrent myocardial infarction(OR=0.32,95%CI 0.11 to 0.95,p =0.04),death,recurrent myocardial infarction,culprit artery restenosis or ventricular arrhythmia(OR=0.59,95%CI 0.36 to 0.96,p=0.03) while these improvements did not reach statistical significance in emergent transfer trials(within 24 hours post AMI).Compared with emergent transfer,BMMNCs therapy at day 4 to 7 also significantly reduced the incidence of revascularization(p for interaction=0.02).
Conclusions
Timing of cell administration might play an important role in the therapeutic response and safety in AMI patients.BMMNCs transfer at day 4 to 7 post AMI was superior to that within 24 hours in improving LVEF,decreasing LVESV and reducing the incidence of revascularization.
PART TWO Time Courses of Myocardial Elasticity,Serum Vascular Endothelial Growth Factor Concentrations and Cardiac Function in Mice with Experimental Myocardial Infarction
Objectives
To investigate the time course of myocardial elasticity,serum vascular endothelial growth factor(VEGF) concentrations and infracted heart function after acute myocardial infarction(AMI) in mice,which makes it possible to take further analyses for the effects of them on the biologic behaviors of transferred cells.
Method
Forty BALB/c mice(6 weeks old,weight 20-25g) were made into murine AMI models by the ligation of left coronary artery and eight sham operations were regarded as the control.Echocardiography and pressure-volume conductance catheter technique were used to evaluate cardiac function and left ventricular pressure at 1 hour,24 hours, 7 days,14 days and 28 days post AMI,respectively.Thereafter serum VEGF concentrations were detected by ELISA technique.Elastic modulus of infarcted myocardium were detected by atomic force microscope,and hematoxylin and eosin and Mallary staining of paraffin section were performed to observe the time-dependent pathologic changes of infarcted heart.Comparisons of continuous variables between two groups were performed by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.
Results
The elastic modulus(E) of myocardium at 1 hour post infarction showed a decrease tendency compared to the normal myocardium(16.60 kPa vs.17.94 kPa,p＞0.05),and the tendency became statistically significant by hour 24 post AMI(E=4.21 kPa, p＜0.001).Thereafter the stiffness of infarcted myocardium gradually increased and had significant differences compared with the three former groups(day 7 post AMI: E=31.38 kPa;day 14:E=53.23 kPa;day 28:E=90.22 kPa;all p＜0.001).In contrast, serum VEGF expression significantly increased by 1 hour after myocardial infarction (49.44 pg/ml vs.38.58 pg/ml,p=0.024) and reached peak concentration at hour 24 (96.30 pg/ml),with statistically significant differences compared to other groups(all p＜0.01).Following the peaking,serum VEGF concentrations gradually decreased and reached normal level by day 14 post AMI(43.89 pg/ml,vs.the control,p＞0.10). Pathological tissue staining showed that,compared with other groups,the injured hearts at hour 24 post AMI had more significant inflammatory cells infiltration following massive myonecrosis.By day 7,the removal of the necrotic myocytes was paralleled by a reduction of inflammatory cells,with the start of immature fibrosis scar formation and complete scar formation by 28 days post infarction.These time-related changes in the above biological and physical parameters were coincidence with those in left ventricular function and pressure.LVEF,FS,and LVESP showed the lowest level at 24 hours post AMI among all the groups(LVEF: 0.50;FS:0.25;LVESP:99.30mmHg;all p＜0.01),meanwhile LVEDP reached the first peak value(12.35 mmHg vs.3.89 mmHg[control group],p＜0.01).
Conclusion:
Elastic modulus of mice myocardium,serum VEGF concentrations and cardiac function after AMI presents time-dependent changes and remains high consistency among them.Elastic modulus of infarcted myocardium experienced a time-dependent process of decreasing at first and then increasing.It reached the bottom at hour 24 post AMI,meanwhile,serum VEGF reached peak concentration and cardiac function remains the worst degrees at the moment.
PART THREE The impacts of matrix elasticity and VEGF on differentiation of bone marrow mononuclear cells to endothelial progenitor cells
Objectives
To investigated the impacts of matrix elasticity and VEGF on specification of bone marrow mononuclear cells(BMMNCs) along endothelial progenitor cells(EPC) by cells culture in the medium with different VEGF concentrations and matrix stiffness similar to the elasticity of infarcted myocardium at different stages of infarction.
Methods
The flexibility of polyacrylamide gel with different acrylamide/bisacrylamide ratios was determined by atomic force microscope to bolt the elastic matrix that could mimick the elastic modulus of infarcted myocardium of hour 1,hour 24,day 7,day 14 and day 28 post AMI.BMMNCs were isolated and cultured in the medium with the above elastic modulus and different VEGF concentrations(0ng/ml,2.5ng/ml,10ng/ml and 20ng/ml).Cellular immunophenotypes(CD133,VEGFR2,CD45 and UEA-1) and uptake of acetylated LDL(ac-LDL),which were specified to EPC,were detected by immunofluorescent technique and flow cytometry.The data based on same elasticity or VEGF concentration were analyzed using one-way ANOVA.The interactive effects of matrix elasticity and VEGF on cell differentiation were measured using two-way ANOVA.The criticalα-level for these analyses was set at p＜0.05. Data analyses were performed by SPSS 11.5 software.
Results
Elastic modulus of the bolted gels was approximately 4kPa,15kPa,42kPa and 72 kPa that could mimick that of infarcted myocardium at hour 24,hour 1,day 7 to 14 and day 14 to 28 post AMI,respectively.Ratio of double positive cells with FITC-UEA-1/DiI-AcLDL had no significant differences among the above flexibility in the culture condition with 10ng/ml to 20ng/ml VEGF(all p＞0.05).With the decrease of VEGF concentrations to 2.5ng/ml in the culture medium,the difference in ratio of double positive cells started to be significant between 42kPa and 15 kPa (72.44%vs.52.44%,p=0.04),and the beneficial effects of 42kPa on promoting cell differentiation became more significant when VEGF concentration was decreased to 0ng/ml.Furthermore,the interactive effect of matrix elasticity and VEGF concentration on stimulating endothelial lineage differentiation was demonstrated by two-way ANOVA(p＜0.01).Regardless of the impacts of VEGF concentrations, 42kPa showed the more favorable effects,whether on absolute numbers or ratio of positive cells,than the others elasticity.Likewise,after controlling the impacts of matrix elasticity,VEGF concentration of 2.5ng/ml seems to be more beneficial both in numbers and in ratio of double positive cells than other concentrations.Moreover, flow cytometry analyses for cells cultured in the medium with 2.5ng/ml VEGF showed that,expression of specified cell surface antigens of EPC still remains the highest level in 42kPa group(CD45(-)/CD133(+)/VEGFR2(+):1.94%).
Conclusions
Matrix elasticity plays an important role on promoting the specification of BMMNCs along EPC.Elastic modulus of 42 kPa,corresponding to of myocardial stiffness at day 7 to 14 post AMI,has a more beneficial effect on pro-differentiation compared with other elasticities.Moreover,an interactive effect on stimulating endothelial lineage differentiation might exist between matrix elasticity and VEGF concentration.
PART FOUR Optimal Timing of Bone Marrow Mononuclear Cells Transplantation for Acute Myocardial Infarction in Mice and the Potential Mechanisms
Objectives
To elucidate the optimal time frame for bone marrow mononuclear cells(BMMNCs) transplantation for acute myocardial infarction(AMI) in mice and the potential mechanisms by detecting capillary density in cell transplantation area and cardiac function after cell injection at different time points post AMI.
Methods
BMMNCs were isolated from BALB/c mice by density gradient centrifugation.AMI animal models were prepared by coronary artery ligation of BALB/c mice.Isolated BMMNCs were directly injected into infarct area at hour 1,hour 24,day 7,day 14 and day 28 post AMI.Injection of M199 medium at corresponding time points was regarded as the control.Two months postinfarct,cardiac function and left ventrieular pressure were measured by echocardiography and pressure-volume conductance catheter technique.Meanwhile,capillary density in the injected area was detected by observing the expression of vWF using immunohistochemical method.Comparisons of continuous variables between cell transfer groups and control groups were performed by independent t test,and comparisons of absolute changes in related parameters between two groups by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.
Results
Capillary density within cell transplantation area in hour 24,day 7 and day 14 injection group was more than the control(control groups:1.32/HP;hour 24,day 7, day 14 injection groups:2.60/HP,4.60/HP,3.80/HP,respectively;all p value＜0.001).Among all the injection groups,day 7 and day 14 injection groups had the highest capillary density.Likewise,the beneficial effects of BMMNCs transplantation at day 7 and day 14 post AMI on absolute changes from control data(△) in LVEF, FS,LVEDP,LVESP and±dp/dt were also demonstrated in present study.The effects of cell transplantation on the above parameters were more favorable in day 7 injection group than hour 24 transfer group(△LVEF:20.95%vs.11.41%,p=0.025;△FS: 13.47%vs.7.13%,p=0.023;△LVEDP:-15.94 mmHg vs.-8.17 mmHg,p＜0.05; +△dp/dt:9001.02mmHg/s vs.4891.53 mmHg/s;p＜0.05),whereas there was no significant differences between day 7 injection group and day 14 injection group (△LVEF:18.82%;△FS:11.63%;△LVEDP:-15.34 mmHg;△LVESP:9.19 mmHg; -△dp/dt:-4650.71 mmHg/s;+△dp/dt:8265.35 mmHg/s;all p value＜0.05).There existed high linear correlations between these parameters(△LVEF,△FS,△LVEDP and△LVESP) and the capillary densities(all r＞0.75,all p＜0.01).Moreover, improvements of LVIDd and LVIDs also showed the more beneficial tendencies in day 7 injection group and day 14 injection group,with no significant differences when compared to the others cell injection groups.
Conclusions
The optimal time frame for BMMNCs therapy for AMI occurs within the period from day 7 to day 14 after the infarction.Cell transplantation at the moment is able to promote the improvements in injured heart function utmost,which might be associated with the highest density of capillary within transplantation area possibly due to the more favorable physical microenvironment(elasticity of infarcted myocardium).
骨髓源细胞移植治疗急性心肌梗死(AMI)的最佳时机问题已引起学术界的广泛关注。已有少量研究表明,移植时机的选择是影响移植疗效的重要因素。目前学术界倾向于最佳移植时机可能出现在AMI后一周左右的观点。然而,其潜在机制尚不明确。
近来越来越多的证据显示,干细胞周围的物理微环境(主要是基质的弹性或硬度,用弹性模量[Elastic Modulus,E]描述)决定着其生物学特性和转归,即由不同弹性基质诱导分化而来的细胞在形态和功能上更接近于宿主组织的细胞。具体来说,类似于脑组织的软基质能够诱导骨髓源干细胞向神经元样细胞分化;类似于肌肉的中等弹性基质能够诱导其发生肌原性分化;类似于骨胶原的硬基质能够刺激其骨原性分化。
同时,考虑到心肌梗死后由大量心肌坏死和细胞外基质的沉积诱发的心肌重构导致梗死心肌发生由软到硬的时间依赖性弹性变化的科学事实,据此,我们提出了AMI后一定时间窗内的心肌弹性可能更有利于移植细胞向有利于心功能改善的方向分化的科学假说,这一时段即为最佳移植时机。
本研究旨在通过一系列研究验证该假说及其前提的正确性,对于这一科学命题的论述将有益于骨髓源细胞移植治疗AMI的时机选择及最佳移植时机潜在机制的阐明。
第一部分移植时间对急性心肌梗死后骨髓单个核细胞移植疗效和安全性影响的荟萃分析
目的通过Meta分析的亚组分析系统评价移植时间对急性心肌梗死(AMI)后骨髓单个核细胞(BMMNCs)移植疗效和安全性的影响。
方法系统检索PubMed、Medline、Cochrane EBM Reviews、EMBASE、BIOSIS等数据库。以AMI症状出现24小时内成功实施经皮冠状动脉介入治疗(PCI)者为研究对象;试验设计体现随机对照原则;移植细胞种类为非动员的自体BMSCs;具有3个月以上的随访资料。样本总量小于10的研究未被纳入。
结果共检出相关文献349篇,筛选出符合纳入标准的文献共9篇,涉及7项研究,总病例数660例。其中心肌梗死后24小时内移植亚组271例,4-7天移植亚组389例,与对照组相比,BMMNCs移植可显著改善左室射血分数(LVEF)和左室收缩末容积(LVESV)。亚组综合效应分析显示,4-7天移植亚组能够明显提高LVEF和减少LVESV(LVEF:权重均差[WMD]=4.63%,95%CI 1.00%-8.26%,p=0.01;LVESV:标化均差[SMD]=—0.28,95%CI—0.53-—0.02,p=0.03),而24小时内移植亚组仅显示出有益趋势,两亚组相比,4-7天移植亚组较24小时内移植亚组可能更有利于LVEF和LVESV的改善,但两亚组间统计学差异无显著性(p＞0.05)。而移植时间对左室舒张末容积(LVEDV)呈中性影响。死亡、支架内或罪犯血管再狭窄、再次心肌梗死、再次血运重建、因心力衰竭再入院等独立不良事件及死亡、再次血运重建、再次心肌梗死或因心力衰竭再入院的联合终点发生率均显示出BMMNCs移植治疗的有益趋势(比值比[OR]均＜1,但p均＞0.05)。与对照组相比,4-7天移植亚组能够明显降低再次血运重建率(OR=0.60,95%CI 0.37-0.97,p=0.04)、死亡或再次心肌梗死的联合发生率(OR=0.32,95%CI 0.11-0.95,p=0.04)及死亡、罪犯血管再狭窄、再次心肌梗死或室性心律失常的联合发生率(OR=0.59,95%CI 0.36-0.96,p=0.03),而这些指标在24小时内移植亚组与对照组间无显著差异;两亚组间相比,4-7天移植亚组的再次血运重建率明显低于24小时内移植亚组(交互p=0.02)。
结论自体BMMNCs移植治疗AMI安全可行,移植时间是移植疗效和安全性的重要影响因素,AMI后4-7天进行细胞移植较24小时内移植其疗效和安全性可能更佳。
第二部分心肌梗死后不同时期心肌弹性、血清VEGF浓度及心脏功能变化
目的探讨小鼠心肌梗死后心肌弹性、血清VEGF浓度和心脏功能的时间变化规律及其潜在联系,为进一步分析心肌弹性和VEGF在骨髓单个核细胞(BMMNCs)分化中的作用和相互作用创造条件。
方法六周龄雄性BALB/c小鼠48只,体重20-25g,采用结扎左冠状动脉制成心肌梗死模型,以假手术动物为对照。分别于心肌梗死后1小时、24小时、7天、14天和28天,行心脏功能和左心室腔内压力检测后,采用ELISA方法检测血清VEGF浓度。每组送检3个标本,应用原子力显微镜检测梗死心肌弹性模量(E);其余心脏组织制成石蜡切片行苏木素-伊红(HE)染色和Mallary纤维染色。多组间两两比较采用单因素方差分析,p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件包进行数理分析。
结果梗死后1小时心肌弹性模量轻微降低(由对照组心肌的17.94 kPa降至16.60 kPa,p＞0.05);24小时后降至最低值(4.21 kPa,p＜0.001),其后梗死心肌弹性逐渐增加,由梗死后7天的31.38 kPa增至4周后的90.22 kPa(与对照组相比,p均＜0.001)。梗死后不同时间点血清VEGF检测显示,心肌梗死后1小时VEGF浓度由正常对照的38.58 pg/ml升至49.44 pg/ml(p=0.024),至24小时达峰值(96.30 pg/ml,p=0.005),心肌梗死后7天、14天和28天逐渐下降(VEGF浓度分别为50.56 pg/ml[p=0.014]、43.89 pg/ml[p＞0.10]、39.82pg/ml[p＞0.10],p值均为与对照组相比)。组织病理染色显示,心肌梗死后24小时,出现大量心肌细胞坏死,炎性细胞浸润与各组相比最为明显;至梗死后7天,胶原纤维逐渐增多,早期疤痕组织开始形成;梗死后14天和28天,纤维疤痕组织逐渐成熟。心肌梗死后心脏功能和左心室腔内压力的改变表现出与上述理化参数相似的变化趋势,AMI后24小时LVEF、FS和LVESP降至最低点(VEGF,0.50对0.86[对照组],p＜0.01;FS,0.25对0.55,p＜0.01;LVESP,99.30 mmHg对144.67 mmHg,p＜0.01),且此时LVEDP亦达到首个峰值(12.35 mmHg对3.89 mmHg[对照组],p＜0.01)。
结论小鼠心肌梗死后心肌弹性模量、血清VEGF浓度和心脏功能均表现出时间依赖性相关变化且相互之间保持较高的一致性。梗死心肌的弹性模量经历了先降低后逐渐增高的变化过程,AMI后24小时心肌弹性模量降至最低,而此时血清VEGF浓度达到峰值;心肌梗死区域炎症反应亦最为明显,且心肌梗死后心脏功能受抑最为明显。
第三部分基质弹性和VEGF在骨髓单个核细胞向内皮祖细胞分化中的作用
目的应用可以模拟梗死后不同时期心肌弹性的弹性可控性细胞培养体系,探讨基质弹性及VEGF在诱导骨髓单个核细胞(BMMNCs)向内皮祖细胞(EPC)分化中的作用和相互作用。
方法采用不同配比浓度的丙烯酰胺和甲叉双丙烯酰胺制成不同硬度的聚丙烯酰胺凝胶;利用原子力显微镜筛选出能模拟心肌梗死后1小时、24小时、7天、14天和28天心肌弹性模量的凝胶用于细胞培养。采用密度梯度离心法分离BMMNCs,每一弹性模量的基质胶上分别采用0ng/ml、2.5ng/ml、10ng/ml、20ng/ml等四个浓度的VEGF进行细胞培养。7天后进行乙酰化低密度脂蛋白(ac-LDL)吞噬/荆豆凝集素-1(UEA-1)结合双阳性细胞免疫荧光检测及EPC表面标志物CD133、CD45、VEGFR2流式细胞分析。同一基质弹性或同一浓度VEGF下的数据分析采用单因素方差分析,基质弹性和VEGF对促细胞分化的相互作用分析应用两因素方差分析,p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件进行数理统计。
结果筛查出4种不同浓度凝胶的弹性模量(分别约为4kPa、15kPa、42 kPa和72kPa)可分别模拟梗死后24小时、1小时、7-14天和14-28天的梗死心肌弹性。FITC-UEA-1/DiI-AcLDL双阳性细胞免疫荧光检测显示,在较高浓度VEGF(10-20ng/ml)培养条件下,贴壁细胞双阳性表达率在各弹性基质间无显著差异(p均＞0.05);当VEGF浓度降至2.5 ng/ml,42kPa基质胶的双阳性表达率显著高于15kPa(72.44%对52.44%,p=0.04),且双阳性细胞数量远大于其他弹性凝胶(p＜0.01);在无VEGF的培养条件下,42kPa在双阳性细胞表达率和细胞数量上表现出较其他弹性更大的优势,此外,4kPa和15kPa双阳性细胞表达率较72kPa显著降低(p均＜0.05)。两因素方差分析显示,基质弹性和VEGF浓度对BMMNCs向EPC的分化存在显著交互作用(p＜0.01),分别控制VEGF浓度和基质弹性的影响后,42kPa的基质弹性和较低浓度的VEGF(2.5-10 ng/ml)较其他弹性和浓度具有更高的的双阳性细胞表达率。此外,流式细胞分析表明,低浓度VEGF(2.5ng/ml)条件下,EPC特征性表面标记物CD45(-)/VEGFR-2(+)/CD133(+)细胞表达率以仍以42 kPa为最高(1.94%)。
结论基质弹性在促进BMMNCs向EPC分化中发挥着重要作用,42 kPa(模拟心肌梗死后7-14天心肌弹性)的促细胞内皮系分化效应优于其他弹性模量。基质弹性与VEGF浓度在促进BMMNCs向EPC分化过程中可能发挥着协同作用。
第四部分心肌梗死后骨髓单个核细胞最佳移植时机及其潜在机制
目的通过对心肌梗死不同时期BMMNCs移植后心脏功能改善状况的研究,探讨心肌梗死后BMMNCs移植的最佳时机及其潜在机制。
方法采用密度梯度离心法分离BALB/c小鼠BMMNCs。分别于心肌梗死后1小时、24小时、7天、14天和28天实施梗死区域BMMNCs局部注射,以相应时间点M199注射为对照,各组于心肌梗死建模后2月,应用小动物超声成像系统和心腔导管压力测试系统对实验动物实施左心功能和左心室腔内压力测定,通过vWF免疫组化方法检测移植区域毛细血管密度。移植组与各对照组比较用成组t检验,多组间两两比较应用单因素方差分析(ANOVA),p＜0.05为差异具有统计学显著意义,应用SPSS 11.5统计软件进行数理统计。
结果与对照组相比,心肌梗死后24小时至14天实施BMMNCs移植可显著提高梗死区域毛细血管密度(对照组:1.32个/高倍镜[HP];AMI后24小时移植组:2.60个/HP;7天移植组:4.60个/HP;14天移植组:3.80个/HP;p均＜0.001),其中以7天移植组的毛细血管密度为最高(两者相比p=0.124)。各移植组LVEF、FS、LVEDP、LVESP及±dp/dt的绝对增量相比,亦显示出相似变化,且以AMI后7天和14天移植组改善最为明显,优于24小时移植(ΔLVEF:20.95%对11.41%,p=0.025;ΔFS:13.47%对7.13%,p=0.023;ΔLVEDP:-15.94 mmHg对-8.17 mmHg,p＜0.05;+Δdp/dt:9001.02mmHg/s对4891.53 mmHg/s,p＜0.05),而与14天移植组相比,统计学差异无显著意义(ΔLVEF:18.82%,p=0.57;ΔFS:11.63%,p=0.46;ΔLVEDP:-15.34 mmHg;ΔLVESP:9.19 mmHg;-Δdp/dt:-4650.71 mmHg/s;+Δdp/dt:8265.35 mmHg/s,p均＜0.05)。ΔLVEF、ΔFS、ΔLVEDP及ΔLVESP等心脏功能相关参数和毛细血管密度间存在高度线性相关(相关系数均＞0.75,p均＜0.01)。此外,7天移植组和14天移植在LVIDd和LVIDs的改善方面呈现的有利趋势亦最为明显,但与其他各组相比差异显著性无统计学意义(p均＞0.05)。
结论AMI后BMMNCs移植治疗的最佳时机出现在心肌梗死后7-14天。在这一时段,细胞移植可最大限度的改善心脏功能和左心室腔内压力,这可能与此时最为适宜的物理微环境(梗死心肌弹性)促进移植区域毛细血管密度的增加进而显著改善了梗死心肌的顺应性有关。
PREFACE
Determining which time point is optimal for bone marrow-derived cells transplantation for acute myocardial infarction(AMI) has attracted a great deal of attention.Studies have verified the interaction between cell treatment effect and transfer timing and have suggested that the optimal time frame for bone marrow-derived cells therapy might be about one week after AMI.
However,the potential mechanism underlying the time-dependent therapeutic response remains unclear.Recently,a growing body of in vitro evidence has suggested that stem cells are able to feel and respond to the stiffness of their microenvironment to commit to a relevant lineage,indicating that soft matrices that mimic brain are neurogenic,stiffer matrices that mimic muscle are myogenic and comparatively rigid matrices that mimic collagenous bone prove osteogenic.
Simultaneously,considering the fact that the myocardium post-infarction experiences a time-dependent stiffness change from flexible to rigid as a result of myocardial remodelling following tissue necrosis and massive extracellular matrix deposition,we presume that the myocardial stiffness within a certain time frame (possibly one week post-AMI) after infarction might provide a more favourable physical microenvironment for the phenotypic plasticity and functional specification of engrafted bone marrow-derived cells committed to some cell lineages,such as endothelial cells,vascular smooth muscle cells or cardiomyocytes.The beneficial effect facilitates angiogenesis and myocardiogenesis in the infarcted heart,and subsequently leads to more amelioration of cardiac functions.
The present study aimed to verify the scientific hypothesis.If the hypothesis were true,it would be of great help to understand the mechanism underlying the optimal timing for bone marrow-derived cells transplantation and to establish a direction for the time selection of cell therapy.
PART ONE Impact of Timing on Efficacy and Safety of Bone Marrow Mononuclear Cells Transplantation in Acute Myocardial Infarction: A Pooled Subgroup Analysis of Randomised Controlled Trials
Objectives
To investigate the impact of timing on efficacy and safety of bone marrow mononuclear cells(BMMNCs) transfer in patients with acute myocardial infarction (AMI) by a pooled subgroup analysis of randomised controlled trials.
Methods
A systematic literature search of PubMed,MEDLINE and Cochrane EBM databases was made on randomised controlled trials with at least 3-month follow-up data for patients with AMI undergoing emergent percutaneous coronary intervention(PCI) and receiving BMMNCs transfer thereafter.
Results
A total of 7 trials with 660 patients were available for analysis.Compared to baseline level,BMMNCs transfer at day 4 to 7 post AMI significantly improved left ventricular ejection fraction(LVEF)(4.63%increase,95%confidence interval[CI] 1.00%to 8.26%,p=0.01),reduced left ventricular end-systolic volumes(LVESV) (95%CI—0.53 to—0.02,p=0.03),and decreased the incidences of revascularization (odd ratio[OR]=0.60,95%CI 0.37 to 0.97,p=0.04) and the cumulative clinical events of death or recurrent myocardial infarction(OR=0.32,95%CI 0.11 to 0.95,p =0.04),death,recurrent myocardial infarction,culprit artery restenosis or ventricular arrhythmia(OR=0.59,95%CI 0.36 to 0.96,p=0.03) while these improvements did not reach statistical significance in emergent transfer trials(within 24 hours post AMI).Compared with emergent transfer,BMMNCs therapy at day 4 to 7 also significantly reduced the incidence of revascularization(p for interaction=0.02).
Conclusions
Timing of cell administration might play an important role in the therapeutic response and safety in AMI patients.BMMNCs transfer at day 4 to 7 post AMI was superior to that within 24 hours in improving LVEF,decreasing LVESV and reducing the incidence of revascularization.
PART TWO Time Courses of Myocardial Elasticity,Serum Vascular Endothelial Growth Factor Concentrations and Cardiac Function in Mice with Experimental Myocardial Infarction
Objectives
To investigate the time course of myocardial elasticity,serum vascular endothelial growth factor(VEGF) concentrations and infracted heart function after acute myocardial infarction(AMI) in mice,which makes it possible to take further analyses for the effects of them on the biologic behaviors of transferred cells.
Method
Forty BALB/c mice(6 weeks old,weight 20-25g) were made into murine AMI models by the ligation of left coronary artery and eight sham operations were regarded as the control.Echocardiography and pressure-volume conductance catheter technique were used to evaluate cardiac function and left ventricular pressure at 1 hour,24 hours, 7 days,14 days and 28 days post AMI,respectively.Thereafter serum VEGF concentrations were detected by ELISA technique.Elastic modulus of infarcted myocardium were detected by atomic force microscope,and hematoxylin and eosin and Mallary staining of paraffin section were performed to observe the time-dependent pathologic changes of infarcted heart.Comparisons of continuous variables between two groups were performed by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.
Results
The elastic modulus(E) of myocardium at 1 hour post infarction showed a decrease tendency compared to the normal myocardium(16.60 kPa vs.17.94 kPa,p＞0.05),and the tendency became statistically significant by hour 24 post AMI(E=4.21 kPa, p＜0.001).Thereafter the stiffness of infarcted myocardium gradually increased and had significant differences compared with the three former groups(day 7 post AMI: E=31.38 kPa;day 14:E=53.23 kPa;day 28:E=90.22 kPa;all p＜0.001).In contrast, serum VEGF expression significantly increased by 1 hour after myocardial infarction (49.44 pg/ml vs.38.58 pg/ml,p=0.024) and reached peak concentration at hour 24 (96.30 pg/ml),with statistically significant differences compared to other groups(all p＜0.01).Following the peaking,serum VEGF concentrations gradually decreased and reached normal level by day 14 post AMI(43.89 pg/ml,vs.the control,p＞0.10). Pathological tissue staining showed that,compared with other groups,the injured hearts at hour 24 post AMI had more significant inflammatory cells infiltration following massive myonecrosis.By day 7,the removal of the necrotic myocytes was paralleled by a reduction of inflammatory cells,with the start of immature fibrosis scar formation and complete scar formation by 28 days post infarction.These time-related changes in the above biological and physical parameters were coincidence with those in left ventricular function and pressure.LVEF,FS,and LVESP showed the lowest level at 24 hours post AMI among all the groups(LVEF: 0.50;FS:0.25;LVESP:99.30mmHg;all p＜0.01),meanwhile LVEDP reached the first peak value(12.35 mmHg vs.3.89 mmHg[control group],p＜0.01).
Conclusion:
Elastic modulus of mice myocardium,serum VEGF concentrations and cardiac function after AMI presents time-dependent changes and remains high consistency among them.Elastic modulus of infarcted myocardium experienced a time-dependent process of decreasing at first and then increasing.It reached the bottom at hour 24 post AMI,meanwhile,serum VEGF reached peak concentration and cardiac function remains the worst degrees at the moment.
PART THREE The impacts of matrix elasticity and VEGF on differentiation of bone marrow mononuclear cells to endothelial progenitor cells
Objectives
To investigated the impacts of matrix elasticity and VEGF on specification of bone marrow mononuclear cells(BMMNCs) along endothelial progenitor cells(EPC) by cells culture in the medium with different VEGF concentrations and matrix stiffness similar to the elasticity of infarcted myocardium at different stages of infarction.
Methods
The flexibility of polyacrylamide gel with different acrylamide/bisacrylamide ratios was determined by atomic force microscope to bolt the elastic matrix that could mimick the elastic modulus of infarcted myocardium of hour 1,hour 24,day 7,day 14 and day 28 post AMI.BMMNCs were isolated and cultured in the medium with the above elastic modulus and different VEGF concentrations(0ng/ml,2.5ng/ml,10ng/ml and 20ng/ml).Cellular immunophenotypes(CD133,VEGFR2,CD45 and UEA-1) and uptake of acetylated LDL(ac-LDL),which were specified to EPC,were detected by immunofluorescent technique and flow cytometry.The data based on same elasticity or VEGF concentration were analyzed using one-way ANOVA.The interactive effects of matrix elasticity and VEGF on cell differentiation were measured using two-way ANOVA.The criticalα-level for these analyses was set at p＜0.05. Data analyses were performed by SPSS 11.5 software.
Results
Elastic modulus of the bolted gels was approximately 4kPa,15kPa,42kPa and 72 kPa that could mimick that of infarcted myocardium at hour 24,hour 1,day 7 to 14 and day 14 to 28 post AMI,respectively.Ratio of double positive cells with FITC-UEA-1/DiI-AcLDL had no significant differences among the above flexibility in the culture condition with 10ng/ml to 20ng/ml VEGF(all p＞0.05).With the decrease of VEGF concentrations to 2.5ng/ml in the culture medium,the difference in ratio of double positive cells started to be significant between 42kPa and 15 kPa (72.44%vs.52.44%,p=0.04),and the beneficial effects of 42kPa on promoting cell differentiation became more significant when VEGF concentration was decreased to 0ng/ml.Furthermore,the interactive effect of matrix elasticity and VEGF concentration on stimulating endothelial lineage differentiation was demonstrated by two-way ANOVA(p＜0.01).Regardless of the impacts of VEGF concentrations, 42kPa showed the more favorable effects,whether on absolute numbers or ratio of positive cells,than the others elasticity.Likewise,after controlling the impacts of matrix elasticity,VEGF concentration of 2.5ng/ml seems to be more beneficial both in numbers and in ratio of double positive cells than other concentrations.Moreover, flow cytometry analyses for cells cultured in the medium with 2.5ng/ml VEGF showed that,expression of specified cell surface antigens of EPC still remains the highest level in 42kPa group(CD45(-)/CD133(+)/VEGFR2(+):1.94%).
Conclusions
Matrix elasticity plays an important role on promoting the specification of BMMNCs along EPC.Elastic modulus of 42 kPa,corresponding to of myocardial stiffness at day 7 to 14 post AMI,has a more beneficial effect on pro-differentiation compared with other elasticities.Moreover,an interactive effect on stimulating endothelial lineage differentiation might exist between matrix elasticity and VEGF concentration.
PART FOUR Optimal Timing of Bone Marrow Mononuclear Cells Transplantation for Acute Myocardial Infarction in Mice and the Potential Mechanisms
Objectives
To elucidate the optimal time frame for bone marrow mononuclear cells(BMMNCs) transplantation for acute myocardial infarction(AMI) in mice and the potential mechanisms by detecting capillary density in cell transplantation area and cardiac function after cell injection at different time points post AMI.
Methods
BMMNCs were isolated from BALB/c mice by density gradient centrifugation.AMI animal models were prepared by coronary artery ligation of BALB/c mice.Isolated BMMNCs were directly injected into infarct area at hour 1,hour 24,day 7,day 14 and day 28 post AMI.Injection of M199 medium at corresponding time points was regarded as the control.Two months postinfarct,cardiac function and left ventrieular pressure were measured by echocardiography and pressure-volume conductance catheter technique.Meanwhile,capillary density in the injected area was detected by observing the expression of vWF using immunohistochemical method.Comparisons of continuous variables between cell transfer groups and control groups were performed by independent t test,and comparisons of absolute changes in related parameters between two groups by one-way ANOVA.The criticalα-level for these analyses was set at p＜0.05.Data analyses were performed by SPSS 11.5 software.
Results
Capillary density within cell transplantation area in hour 24,day 7 and day 14 injection group was more than the control(control groups:1.32/HP;hour 24,day 7, day 14 injection groups:2.60/HP,4.60/HP,3.80/HP,respectively;all p value＜0.001).Among all the injection groups,day 7 and day 14 injection groups had the highest capillary density.Likewise,the beneficial effects of BMMNCs transplantation at day 7 and day 14 post AMI on absolute changes from control data(△) in LVEF, FS,LVEDP,LVESP and±dp/dt were also demonstrated in present study.The effects of cell transplantation on the above parameters were more favorable in day 7 injection group than hour 24 transfer group(△LVEF:20.95%vs.11.41%,p=0.025;△FS: 13.47%vs.7.13%,p=0.023;△LVEDP:-15.94 mmHg vs.-8.17 mmHg,p＜0.05; +△dp/dt:9001.02mmHg/s vs.4891.53 mmHg/s;p＜0.05),whereas there was no significant differences between day 7 injection group and day 14 injection group (△LVEF:18.82%;△FS:11.63%;△LVEDP:-15.34 mmHg;△LVESP:9.19 mmHg; -△dp/dt:-4650.71 mmHg/s;+△dp/dt:8265.35 mmHg/s;all p value＜0.05).There existed high linear correlations between these parameters(△LVEF,△FS,△LVEDP and△LVESP) and the capillary densities(all r＞0.75,all p＜0.01).Moreover, improvements of LVIDd and LVIDs also showed the more beneficial tendencies in day 7 injection group and day 14 injection group,with no significant differences when compared to the others cell injection groups.
Conclusions
The optimal time frame for BMMNCs therapy for AMI occurs within the period from day 7 to day 14 after the infarction.Cell transplantation at the moment is able to promote the improvements in injured heart function utmost,which might be associated with the highest density of capillary within transplantation area possibly due to the more favorable physical microenvironment(elasticity of infarcted myocardium).
引文
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