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磁标记间充质干细胞治疗急性心肌梗死的实验研究
Magnetically-labeled Mesenchymal Stem Cells Implantation in Ischemic Swine Heart: A Magnetic Resonance Imaging Investigation
【作者】 黄浙勇；
【作者基本信息】 复旦大学 ， 心血管内科学， 2008， 博士
【摘要】 大量动物和临床试验表明骨髓干细胞移植能有效改善缺血心脏功能,但评价细胞植入、分布、存活、迁移的细胞标记示踪研究明显滞后。利用磁共振（MR）活体示踪技术评价心肌梗死干细胞治疗的研究目前尚处于起始阶段,尚存在许多亟待解决的问题。本研究立足于心脏病细胞治疗中的示踪热点,在优化了猪急性心肌梗死模型的基础上,采用超顺磁性氧化铁纳米颗粒Resovist标记猪骨髓间充质干细胞（MSCs）;对磁标MSCs的体外、心肌内移植MR影像学特点、活体长期MR示踪可行性进行了详细研究。体外研究结果发现,Resovist可高效安全标记细胞,MR信号能一定程度上反映磁标记细胞的数量,Resovist颗粒在细胞内停留时间约为2周。动物实验发现,磁共振成像对活体心肌内移植的磁标细胞具定位、相对定量价值,但磁共振无法区分细胞内外铁颗粒;磁共振长期活体细胞示踪并不能反映细胞转归,可能与心肌组织无法及时清除磁标细胞释放的间质铁颗粒有关。量效关系研究发现,间充质干细胞局部移植治疗猪心肌梗死的疗效呈剂量依赖性。第一部分冠脉结扎法制作猪急性心肌梗死模型的优化目的:（1）比较前降支结扎坐标（对角支vs.LAD百分位点）对猪心肌梗死模型稳定性的影响,以期指导研究者根据模型的心功能要求选择合适的LAD结扎位置;（2）探讨前降支结扎后发生室颤的特点及其相关因素,提高动物成活率。方法:（1）47头小型猪随机分为两组:A组（n=24）结扎LAD第二对角支开口远端;B组（n=23）结扎LAD中远1/3交界处。分别于术前、术后1小时心脏超声检查左室射血分数（LVEF）,术后3天进行常规冠脉造影,4周处死测量LAD结扎位点和梗死体积,并比较两组间各指标变异程度,用简单直线回归模型分析LAD结扎百分位点和心梗体积、LVEF相关性和回归方程。（2）57只小型猪开胸结扎心脏LAD不同位点,对室颤和体重、性别、术前心率、术前LVEF、开胸径路（旁正中/肋间）、手术时间、结扎百分位点、术后心率、术后发生室早或短阵室速等因素进行单因素相关分析和Logistic回归分析。结果:（1）B组结扎位点距离LAD开口长度、梗死体积、术后LVEF的变异较小;冠脉造影示对角支的开口、直径和数量存在很大变异;LAD结扎百分位点和梗死心肌体积、术后LVEF均明显相关;线性回归方程分别为:心肌梗死体积（%）=-0.54×结扎百分位点+48.06;术后LVEF（%）=0.56×结扎百分位点+10.49。（2）室颤均发生在结扎冠脉后35 min内,高峰时间为结扎冠脉后5 min和20 min;心率快于160 bpm或慢于60 bpm时容易诱发室颤。与非室颤组动物比较,室颤组动物的结扎位点高,术后最快心率＞60 bpm的动物较多,短阵室速发生率高。Logistic回归分析显示结扎位点过高是急性心肌梗死后发生室颤唯一的独立危险因素。结论:（1）由于对角支变异较大,以LAD全长为坐标选择合适的结扎百分位点可能是一种较为理想的猪心肌梗死模型建立方法。（2）结扎位点过高是猪急性心肌梗死后发生室颤的最重要危险因素;冠脉结扎后30min内应该严密心电监护,特别注意结扎冠脉后5 min和20 min二个时间点、＞160 bpm或＜60 bpm二种心率、以及短阵室速等先兆事件。第二部分猪间充质干细胞的磁化标记和体外MR成像目的:确定超顺磁性氧化铁纳米颗粒Resovist标记猪骨髓间充质干细胞的合适方案,探讨磁标干细胞的体外MR成像特点、影响因素和铁衰减特点。方法:分离培养鉴定猪骨髓间充质干细胞后,首先用不同Resovist浓度、不同共孵育时间标记,根据标记效率指标（普鲁士蓝染色、细胞铁浓度测定、细胞透射电镜检查）和标记毒性指标（台盼蓝排除试验、CCK-8试验）确定适宜标记条件。然后收集不同数量标记细胞重悬于琼脂糖EP管或将细胞沉淀注入凝胶,进行1.5 T MR仪不同序列体外成像,分析体外铁标细胞的MR成像特点和检测阈值。最后收集标记后不同时间点细胞进行普鲁士蓝染色、细胞内铁离子浓度测定和MR成像,分析细胞内铁颗粒衰减特点。结果:（1）权衡标记效率和标记毒性,“25:0.75μg/ml Resovist-PLL 24h标记方案”和“50:1.5μg/ml Resovist-PLL 24h标记方案”是比较合适的标记策略。此二种方案标记后,细胞内铁平均含量为（13.13±2.30）pg和（23.77±2.06）pg,普鲁士蓝染标记率接近100%,电镜检查显示胞质内含铁致密颗粒囊泡,细胞微细结构未见破坏和毒性作用。（2）相比FSE T2WI和FSE T1WI,T2*WI flash信号改变最为明显;成像细胞数量越多,信号改变越明显。临床用1.5 T MR成像仪体外MR T2*WI flash检测阈值为5×104～1×105个细胞。（3）随着标记后体外培养时间延长,细胞内铁逐渐减少,16 d时铁含量接近标记前水平,12 d MR信号强度和标记前无差异。结论:“25:0.75μg/ml Resovist-PLL 24h标记方案”和“50:1.5μg/mlResovist-PLL 24h标记方案”可高效标记猪MSCs;体外MR信号强度能一定程度上反映磁标记细胞的数量;Resovist颗粒可在体外MSCs细胞内停留2周左右时间。第三部分磁标记猪间充质干细胞心肌内移植的体内MR成像目的:探讨活体心肌内磁标干细胞MR成像的特点和影响因素,研究MR成像对标记细胞的定性、定量和定位价值。方法:“50:1.5μg/ml Resovist-PLL 24h标记方案”磁化标记猪骨髓MSCs,并用DAPI和PKH26进行荧光标记。然后将MSCs经心外膜直接注入猪急性梗死心肌内,每头猪注射3点。根据每注射点细胞数量以及细胞是否铁标记,15头猪随机分成5组（每组n=3）:2×106标记细胞组、1×106标记细胞组、5×105标记细胞组、1×106未标记细胞组和单纯SPIO组。细胞移植后20～24h行猪心脏1.5T MR成像,包括快速自旋回波（FSE）T1WI和T2WI序列、快速小角度反转梯度回波T2WI-flash、T2-MAP以及注射造影剂后的T2WI-flash。1h后处死并根据MRI图像切取心肌组织行普鲁士蓝染色和免疫荧光检查。结果:（1）体外标记:铁标记率为100%,DAPI和PKH26共阳性率达（93±5）%。（2）MRI:未标记细胞组未显示低信号区;单纯SPIO组和标记细胞各组均可显示低信号区。相比TrueFrsp序列、T1WI-FSE和T2WI-FSE序列,T2WI-flash序列能更清晰的显示移植的磁标记细胞;T2-MAP序列成像和钆喷酸葡胺注射后T2WI-flash序列成像,图像质量优于单纯T2WI-flash序列成像。可有效避免心电门控欠佳和呼吸心跳频率影响。T2WI-flash结合TrueFrsp序列（或T1WI-FSE或T2WI-FSE序列）,或者钆喷酸葡胺注射后T2WI-flash序列,或者采用T2-MAP成像序列,MR可判断操作成功率。心脏T2WI-flash成像时,心肌内低信号区面积和回波时间呈线性相关。临床用1.5TMRI仪至少可以探测到活体心脏内5×105的细胞数量。在回波时间相近或相同的情况下,注射点低信号区的面积大小与移植细胞数量显著正相关。（3）组织学检查:各标记细胞组动物MRI低信号区心肌病理学检查见普鲁士蓝染色、DAPI和PKH26三重阳性细胞存在;无SPIO标记细胞组注射部位心肌中存在DAPI和PKH26阳性细胞,单纯SPIO组心肌注射点显示细胞外游离的铁颗粒。结论:应用磁共振对超顺磁性氧化铁标记的干细胞进行活体心肌内成像是可行的,具定位、相对定量价值,但无法区分细胞内、外铁颗粒。第四部分磁标记间充质干细胞治疗急性心肌梗死的磁共振长期示踪和量效关系研究目的:量效关系是细胞移植治疗心肌梗死疗效评价中必不可少的组成部分,但文献较少。细胞磁共振成像能否长期示踪并真实反映移植细胞的迁移尚不清楚。本研究用MRI监测不同剂量的磁标MSCs移植治疗猪急性心肌梗死,观察移植细胞剂量和移植疗效的关系,并探讨MRI在示踪干细胞中的价值。方法:开胸结扎冠状动脉LAD建立猪急性心肌梗死模型,将磁标MSCs经心外膜直接注入梗死交界区心肌内,每头猪注射10点。根据移植细胞数量,15头猪随机分成3组:高剂量组（移植细胞量2×107,n=5）、中等剂量组（移植细胞量1×107,n=5）、低剂量组（移植细胞量5×106,n=5）。对照组仅注射DMEM（n=5）。细胞移植后24和1、3、6个月行猪心脏1.5T MR成像,采集心脏功能、测定梗死面积、移植细胞信号改变等信息。6个月处死,梗死区和梗死交界区切片进行HE染色、Mallory染色观察组织细胞形态和纤维化情况;Ⅷ因子免疫荧光评价新生血管情况;Tunel法免疫组化观察组织细胞凋亡情况;普鲁士蓝染色和CD68免疫组化观察铁颗粒状态和心肌细胞、巨噬细胞的关系;结果:（1）磁共振心功能检查发现,对照组和低、中、高剂量组基线LVEF平均值分别为29.96%±1.45%、30.04%±2.20%、29.80%±2.82%和29.04%±1.60%。与对照组比较,移植3个月后低、中和高剂量组分别增加0.12%、1.78%、3.72%和6.68%,随访6个月后分别增加-1.08%、1.82%、4.10%和7.24%。重复测量分析显示心功能改善随细胞剂量不同而不同,细胞剂量和心功能改善幅度相关。（2）细胞磁共振活体示踪发现基本演变趋势为:移植后3d呈边界清晰的低信号;随后边界模糊,面积扩大,与正常交界的心内膜缘呈低信号;注射点对比度下降,并逐渐隐退或消失。组间比较发现剂量越高,显示率越高,信号示踪时间越长,细胞移行现象越明显。（3）磁共振测量梗死面积发现,对照组和低、中、高剂量组的基线R心梗均值分别为18.50%±1.94%、19.78%±1.43%、18.96%±1.33%和19.48%±1.12%;与对照组比较,移植3个月低、中和高剂量组分别减少0.86%、3.16%、3.52%和5.24%,随访6个月后分别减少1.12%、3.72%、4.62%和6%。重复测量分析显示,细胞剂量和梗死面积变化相关。（4）新生血管密度与移植细胞数量密切相关,梗死周边区低、中和高剂量组新生血管密度分别是对照组的1.35倍、2.89倍和4.71倍;梗死区低、中和高剂量组分别是对照组的1.23倍、2.36倍、4.18倍。（5）梗死以及梗死边缘区凋亡指数（AI）,对照组为4.2±1.0,低、中、高剂量组均显著低于对照组,分别为2.4±1.7、1.3±1.2、0.8±1.1（P均＜0.05）;有“剂量越大、凋亡越少”的趋势,提示移植细胞不仅减少凋亡,与移植细胞数量可能相关。（6）普鲁士蓝染色和CD68免疫组化:绝大部分铁颗粒游离于细胞之外,少数含铁细胞不符合心肌细胞或巨噬细胞的特征。对照组心肌壁内未显示普鲁士蓝染颗粒。结论:骨髓间充质干细胞局部移植治疗急性心肌梗死疗效呈剂量依赖性特点,但MRI长期活体细胞示踪可能并不能反映细胞转归。
【Abstract】 PART ONE:Optimization of open-chest myocardial infarction model of swineObjectiveDifferent occlusion sites of left anterior descending artery（LAD） have been used for establishing model of myocardial infarction（MI） in pigs.However,little is known about the correlation between the LAD ligating positions and the quality of MI model. The aims of this study are:①to compare the characteristics,stability in particular,of acute myocardial infarction swine model by two different procedures:ligation just below D2 branch,or ligation at one-third distal section of LAD.And to explore the correlation among site of LAD ligation,infarct size,and left ventricular function.②to explore the characteristics and risk factors of ventricular fibrillation（Vf） in open-chest acute myocardial infarction models of swine.Methods①Forty-seven miniswines were divided into group A（ligation just below D2 branch,n=24） and group B（ligation at one-third distal section of LAD,n=23）.Left ventricular ejection fraction（LVEF） was measured by echocardiography before and after MI.Coronary angiography was used for confirming the ligation position after MI and the histological ischemic area was examined at 4 weeks after MI.The variations of each parameter were compared between the two groups.Linear regression analysis was used to analyze the correlation between LAD ligation position and infarct size as well as LVEF.②Forty-seven miniswines mentioned before,along with another 10 miniswines underwent same surgery were divided into Vf group and Vf-free group based on the occurrence of Vf during procedure.The weight,gender,heart rate（HR） before and after MI,LVEF,thoracotomy route（intercostal or parasternal）,operation duration, percentage point of LAD ligation,arrhythmia after MI were recorded and compared between Vf group and Vf-free group.The risk factors of Vf were determined by logistic regression analysis.ResultsThirty-nine pigs survived the operation.Ischemic area was significantly smaller, post MI LVEF was significantly higher and the variations of these parameters were significantly smaller in group B（n=20） than that in group A（n=19）.The difficulty in identifying ventricular branches,along with great variation of branching pattern, encumbers the precise ligation in group A.Eighteen of the 57 pigs suffered from ventricular fibrillation within 35 min after LAD ligation and 11 cases were died.Compared with Vf-free group,Vf group featured a higher position of LAD ligation,a higher incidence of ventricular tachycardia and maximal HR＞160 bpm post MI.Logistic regression analysis showed the position of LAD ligation was the sole risk factors of Vf after MI with statistical significance.Pigs with HR＞160 bpm or＜60 bpm post MI was susceptible to Vf.Conclusion①Ligating LAD with its whole length rather than ventricular branch as coordinate may be more practical and advisable in establishing reproducible MI models,allowing for standardization the location of occlusion and infarct size.②The position of LAD ligation is the vital determinant of Vf in establishing acute MI model of swine.Attentions should be paid to the first 30 min after LAD ligation, HR,and ventricular tachycardia.PART TWO:Labeling of swine bone marrow mesenchymal stem cells with Resovist and MR imaging In vitroObjectiveTo label swine bone marrow mesenchymal stem cells（MSCs） with Resovist combined with poly-1-lysine（PLL）,and to determine the characteristics of magnetically-labeled stem cells with MR imaging in vitro.MethodsSwine mesenchymal stem cells were isolated,culture-expanded 3 passages,and identified by Fluorescence-activated Cell Sorting（FACS） in vitro.MSCs were magnetically labeled with different concentration of Resovist in culture medium for different incubation duration,so as to determine the appropriate labeling methods according to both cellular iron incorporation and cell toxity.Accumulation of particles of SPIO in MSC was assessed by Prussian Blue staining,atomic absorption spectrometry and electron microscopy.Cell toxity were determined by trypan-blue exclusion test,CCK-8 test and electron microscopy.Magnetically-labeled stem cells suspensions underwent MR imaging with T1-weighted spin-echo,T2-weighted fast field-echo and T2-weighted flash sequences at a clinical 1.5 T MR system to determine the characteristics of MR imaging in vitro.The attenuation of Resovist within the labeled cells was assessed by Prussian Blue staining,atomic absorption spectrometry and MR imaging.ResultsLabeling MSCs with 25μg/ml or 50μg/ml of Resovist in culture medium for 24 hours is appropriate.Intracytoplasmic particles stained with Prussian blue stain were detected for all cells with mean cellular iron content of（13.13±2.30） pg per cell for 25μg/ml Resovist in medium,and（23.77±2.06） pg per cell for 50μg/ml Resovist in medium.In vitro MR images showed signal intensity changed most obviouly in T2-weighted flash sequence.The percentage change of signal intensity increased with increasing cell numbers.As few as 5×104-1×105 cells could be detected using this approach.With division of stem cells,the plasma iron decreased gradually,and approached the prelabeling baseline level 12 to 16 days after labeling.ConclusionSwine bone marrow mesenchymal stem cells can be successfully and efficiently labeled with Resovist and can be depicted with a standard 1.5-T MR imager in vitro. The incorporated Resovist particles could retain within cells for 2 weeks.PART THREE:In vivo cardiac magnetic resonance Imaging of Resovist-labeled mesenchymal stem cells in swinesObjectiveTo explore the feasibility and value of magnetic resonance imaging（MRI） of transplanted Resovist-labeled MSCs in swine heart with acute MI.MethodsMSCs were isolated from miniswines.After 24-hour incubation with nanometer Resovist,the third-passage MSCs was further labeled with DNA dye 4’-6-diamidino-2-phenylindole（DAPI） and aliphatic red fluorescent dye PKH26-GL. Approximately 1 hour following the ligation of left anterior descending coronary artery in 20 swines,SPIO-labeled MSCs（5×105 per 150μl injection,n=3;1×106 per 150μl injection,n=3;and 2×106 per 150μl injection,n=3）,MSCs without SPIO （1×106 per 150μl injection,n=3）,and SPIO without MSCs（2μl Resovist per 150μl injection,n=3） were transextracardially implanted into the freshly infarcted myocardium.Each swine received 3 injections.All 20 miniswines were performed MR imaging with T1-weighted spin-echo,T2-weighted fast field-echo,T2-weighted flash,T2-MAP,and T2-weighted flash sequence after the intravenous injection of magnevist at a clinical 1.5 T MR system 20 to 24 hours post infarction.All 20 miniswines subsequently sacrificed for histology just atl hour post MRI.ResultsNo areas of low signal were identified in the SPIO-free cell transplanted hearts.All injected sites containing SPIO-MSCs,as well as SPIO without MSCs were detected in vivo in a clinical 1.5T scanner,presenting low signal intensity spots with the scanning T2-weighted flash sequence,while obscure or even invisible with T1-weighted spin-echo and T2-weighted fast field-echo sequences.The quality of MR image with T2-MAP or T2-weighted flash sequence after the intravenous injection of magnevist was superior to T2-weighted flash.The area of low signal with T2-MAP is linearly correlated to echo time and cell number transplanted.As few as 5×105 cells could be detected in a clinical 1.5T scanner.Prussian blue positive,as well as DAPI and PKH26 fluorescently labeled cells were detected on histology in sections corresponding to lesions shown on MRI in animals transplanted with SPIO-labeled MSCs.ConclusionMagnetically labeled MSCs transplanted in swine myocardial ischemia can be visualized in vivo with MRI at clinical 1.5-T field strength,suggesting the feasibility of tracking SPIO-MSC clinically.PART FOUR:Dose-effect relationship of magnetically-labeled mesenchymal stem cells in swines with acute myocardial infarction: MRI assessmentObjectiveThere is a paucity of information correlating the number of transplanted stem cell after myocardial infarction（MI） with the beneficial effect on infracted heart. Therefore,we assessed the dose-effect relationship of transplanted magnetically-labeled mesenchymal stem cells（MSCs） in swines with acute MI using magnetic resonance imaging（MRI）.MethodsAfter 24-hour incubation with nanometer Resovist and poly-1-lysine（PLL）,MSCs were transextracardially implanted into the freshly infarcted myocardium approximately 1 hour following the ligation of LAD in 15 swines.Swines were randomized into 3 groups according to the number of MSCs:low dose group（n=5）, 5×106;medium dose group（n=5）,1×106;high dose group（n=5）,2×107.Another 5 swines were received DMEM medium only as control group.The dynamic changes of transplanted cells,infarct area and ventricular function were monitored by 1.5-T clinical MRI at 24 hours,1,3 and 6 months post infarction.All 20 miniswines subsequently sacrificed for histology at months post infarction.Immunohistochemical staining with antibodies against CD68 was performed to show the distribution of macrophage.The number of blood vessels was identified by the expression of yon Willebrand factor.The apoptosis was assessed by Tunel immunohistochemical staining.HE staining,Mallory staining and Prussian Blue staining were also performed.ResultsThe improvement of LVEF was obviously related to the number of transplanted MSCs 3 months after transplantation[0.12%in control group,1.78%in low dose group,3.72%in medium dose group（P＜0.05,compared with control）,6.68%in high dose group（P＜0.05,compared with control）,respectively],as well as 6 months after transplantation[-1.08%in control group,1.82%in low dose group,4.10%in medium dose group（P＜0.05,compared with control）,7.24%in high dose group（P＜0.05, compared with control）,respectively].The changes of infarct area,the number of blood vessels and the apoptosis index are also related to the number of transplanted MSCs.SPIO-labeled MSCs presenting low signal intensity spots with the scanning T2*WI-Flash2d sequence.The duration of discernable spots and the migration extent of the sports were related to the dose of injected cells.Histology showed the majority of iron particles were extracellularly distributed.ConclusionsLocally injected MSCs in infracted myocardium can improve ventricular function in a dose-dependent manner.The persistence of significant iron-dependent MRI signal derived from extracellular Resovist indicates that MRI of Resovist-labeled cells does not reliably report long-term stem cell engraftment in the heart.