磁共振活体示踪猪心梗后经冠脉移植间充质干细胞实验研究
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摘要
研究背景
骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cellsMSCs)移植可以修复受损心肌细胞、促使心肌细胞再生、改善心脏功能。MSCs移植已经成为一种新的方法用于心肌细胞再生替代治疗,为临床治疗心血管疾病开辟了一个新的领域。为了明确细胞移植后,脏器功能的改善是否确由移植细胞所引起,需要知道移植细胞能否到达心肌梗塞区域,能否在心肌梗塞区域存活。而观察移植干细胞在体内迁移和转归一直是让人困扰的问题。既往研究多通过处死实验动物行组织切片检查加以证实,这种方法不利于活体对移植细胞迁移做动态观察,也无法适用于临床研究。因此,若将干细胞移植真正应用于临床治疗,必须解决在活体如何识别、追踪移植的干细胞。本研究用超顺磁性氧化铁纳米粒子(superparamagneticiron oxide,SPIO)及CM-DiI荧光双标记骨髓间充质干细胞;通过微创法建立小型猪闭胸式心肌梗塞(myocardial infarction MI)模型;用Over-The-Wire(OTW)球囊经冠脉移植SPIO及CM-DiI双标记的骨髓间充质干细胞;通过磁共振分子成像来活体示踪移植的骨髓间充质干细胞在心肌中的分布。而且本研究还根据磁共振活体示踪结果,创新性的借用分选造血干细胞及肿瘤干细胞的方法:荧光激活细胞分离技术(fluorescence-activated cell sorting,FACS)分选出移植到心肌梗塞区域的骨髓间充质干细胞,为进一部研究骨髓间充质干细胞的作用机理奠定基础。
第一部分超顺磁性氧化铁纳米离子和CM-DiI双标记骨髓间充质干细胞
目的:观察SPIO及CM-DiI对骨髓间充质干细胞的标记效果。方法:全骨髓及密度梯度离心法分离培养猪MSCs。50μg/mL的SPIO及CM-DiI标记第3代MSCs,普鲁士蓝铁染色鉴定SPIO的标记效率,电子显微镜观察细胞内SPIO的摄取;荧光显微镜下观察CM-DiI标记效率。台盼蓝排斥实验观察标记后细胞活性,四唑盐(MTT)比色实验观察标记后细胞生长情况。结果:SPIO及CM-DiI标记MSCs效率高SPIO浓度为50μg/mL,标记时间24h对MSCs的生长、增殖无影响。结论:SPIO及CM-DiI标记猪骨髓间充质干细胞效率高,对细胞生长增殖无影响。
第二部分建立猪闭胸式心肌梗塞模型
目的:用微创法建立猪闭胸式心肌梗塞模型。方法:球囊封堵位置在第一对角支远端与第二对角支近端之间,封堵时间为60min。术中心电监护持续监测心电图变化,球囊封堵结束后冠脉造影观察远端血流中断情况。造模7周后行心肌组织病理学检查。结果:造模手术的27头猪死亡3头,24头成功建立心肌梗塞模型;球囊封堵结束后冠脉造影示前降支中远端血流中断;心肌组织病理切片示梗塞区心肌坏死完全。结论:微创法建立猪闭胸式心肌梗塞模型是一种可行、实用的心梗模型制作方法。
第三部分经冠脉移植SPIO及CM-DiI双标记骨髓间充质干细胞
目的:评价心肌梗塞后经冠脉移植骨髓间充质干细胞的效果及安全性。方法:27头猪分为5组:MI移植双标记MSCs组(n=6);MI移植SPIO加PBS组(n=6);MI移植CM-DiI单标记MSCs组(n=6);MI对照组(n=6);健康猪移植双标记MSCs组(n=3)。心肌梗塞模型建立2周后每头猪经冠脉移植1×10~8个细胞。细胞移植后5周行超声心动图检查,比较MI移植双标记MSCs组及MI对照组左室射血分数及短轴缩短率。心肌组织切片行荧光显微镜观察及普鲁士蓝铁染色,观察经冠脉移植MSCs的效果。结果:实验动物均成功完成MSCs移植,细胞移植中动物无死亡、未见ST段抬高及严重心律失常。细胞移植后5周,MI移植双标记MSCs组射血分数及短轴缩短率高于MI对照组(P＜0.05)。普鲁士蓝铁染色示移植的MSCs位于心肌梗塞区域,呈蓝色。心肌组织冰冻切片荧光显微镜观察移植的MSCs发出红色荧光,呈条带状分布。结论:经冠脉移植SPIO及CM-DiI双标记骨髓间充质干细胞是安全、有效。
第四部分磁共振活体示踪心肌内移植的骨髓间充质干细胞
目的:观察磁共振能否活体示踪到经冠脉移植的骨髓间充质干细胞。方法:SPIO标记猪MSCs,普鲁士蓝铁染色鉴定SPIO标记效率。SPIO标记的MSCs在体外行细胞水平磁共振成像。经冠脉移植骨髓间充质干细胞第1、3、5周后行猪心脏磁共振检查。采用快速梯度回波序列完成长轴位四腔心和二腔心扫描,在以长轴位四腔心和二腔心为定位相,垂直于室间隔获得左心室短轴位图像。根据磁共振活体示踪结果,从心尖向心底把心脏切成6～8片。心肌组织切片行普鲁士蓝铁染色及荧光显微镜观察,验证磁共振活体示踪结果。结果:经冠脉移植SPIO及CM-DiI标记的骨髓间充质干细胞在磁共振成像上显影,表现为低信号影,随着时间推移,低信号影逐渐减弱,并可持续至移植后5周。磁共振活体示踪结果与病理组织学检查结果一致。结论:证明磁共振能活体示踪到经冠脉移植的骨髓间充质干细胞。
第五部分荧光激活细胞分离技术分选出心梗区移植的骨髓间充质干细胞
目的:探讨荧光激活细胞分离技术从心肌梗塞区分选移植的MSCs可行性。方法:制备新鲜心肌组织单细胞悬液,用流式细胞仪(EPIC ALTRA,BeckmanCoulter)F/1通道分选,激发波长:488nm。分选出CM-DiI标记的移植到心肌梗塞区的骨髓间充质干细胞。荧光显微镜观察分选出的移植细胞,流式细胞仪检测分选出的移植细胞周期,并与移植前的MSCs比较。结果:荧光激活细胞分离技术(FACS)从心肌梗塞区分选出移植的骨髓间充质干细胞,进一步证明了磁共振活体示踪结果。分选出的移植细胞荧光显微镜下观察:细胞呈圆形,细胞均显红色荧光,与移植前的细胞类似。分选出的移植细胞周期测定结果示:G1期为58.3%,增殖指数G2+S期达41.6%。结论:荧光激活细胞分离技术(FACS)能从心肌组织分选出移植的MSCs,该方法为干细胞示踪研究提供新的思路。
Background
Coronary artery occlusion leads to ischemia and cell death in the heart. Cardiomyocyte death results in scar formation and left ventricular dilatation ultimately leads to progressive heart failure.
Bone mesenchymal stem cells(MSCs) transplantation provides a potential regenerative therapy for the heart damaged by myocardial infarction. The mechanism of MSCs transplantation therapy remains unclear. Numerous studies have been undertaken in animals and humans to analyze the efficacy of this new approach. The success of cell therapy will depend on the ability to monitor the fate of transplanted cells in vivo. However, rare studies evaluated of survival, migration, and differentiation status of transplantation MSCs. Many methods have been developed to track the destination of the injected stem cells. These tracking methods only confirm the presence of transplantion stem cells in postmortem recipient tissues .
To better understand the mechanism of cell therapy and potential beneficial effects observed in clinical trials. There is a need to track the transplanted stem cells in vivo.
Recent advances in the field of MR contrast media and cell labeling supports molecular and cellular imaging. One such approach involves the use of superparamagnetic iron oxide (SPIO) particles as a contrast agent for cell tracking. This exciting new area offers the potential for non-invasive tracking of implanted cells, ideal for monitoring in the clinical setting. The present study including: superparamagnetic iron oxide (SPIO) particles and fluorescent CM-DiI dye dual labeled MSCs;a minimal-invasive model of myocardial infarction in swine; intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarct model; in vivo magnetic resonance imaging tracking of bone marrow-derived mesenchymal stem cells via intracoronary administration; according to MR findings, sorted the fluorescently labeled transplantation cells from injury myocardium using fluorescence-activated cell sorting (FACS), a potential novel technique for tracking of transplantation cells.
Part I Superparamagnetic iron oxide particles and fluorescent CM-DiI dye dual labeled MSCs
Objective: To investigate the effects of labeling MSCs with SPIO and CM-DiI.Methods: Porcine MSCs were isolated and cultured by the whole bone marrow method and by Percoll density gradient centrifugation. MSCs were labeled with 50μg/mL SPIO and fluorescent CM-DiI dye. The labeling efficiency was tested through Prussian blue staining and fluorescent imaging.The intracellular iron uptake was also assessed with electron microscopy. Labeled MSCs viability and proliferation were determined using Trypan blue rejection method and MTT. Result: Nearly 100% of the MSCs were labeled with SPIO and CM-DiI . SPIO at 50μg/mL doses and treatment times of 24 h did not statistically affect the viability and proliferation of MSCs.Conclusion: Porcine MSCs could be efficiently and safely labeled with SPIO and CM-DiI.
Part II A minimal-invasive model of myocardial infarction in swine
Objective: To develop a closed-chest, minimal-invasive swine model of myocardial infarction.Methods: A balloon catheter was advanced into the left descending coronary artery directly beyond the second diagonal branch. The balloon was inflated and occlusion of the vessel, Angiography confirmed while ECG was continuously monitored. The balloon was deflated after 60min.Result: Myocardial infarction was successfully induced in 24 animals. After balloon occlusion, coronary angiography shows blood flow interruption in the distal left anterior descending branch.Post-mortem histological analysis revealed myocardial necrosis.Conclusion:The closed chest,minimally invasive methods represent a useful alternative for studies of myocardial infarction.
Part III Intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarc model
Objective: To evaluate the effects and safety of intracoronary infusion of mesenchymal stem cells after myocardial infarction.Methods: A total of 27 swine were divided into five groups:healthy animals with dual-labeled MSCs transplantation group(n=3);MI control group(n=6);MI with dual-labeled MSCs transplantation group(n=6); MI with SPIO and PBS transplantation group(n=6); MI with CM-DiI labeled MSCs group(n=6). 1×10~8 MSCs were delivered for one model by intracoronary injection 2 week post myocardial infarction. Heart function were observed 5 weeks after transplantation.The transplantation efficiency was tested through Prussian blue staining and fluorescent imaging.Result: During intracoronary injection of dual labeled MSCs, no adverse events were noted. Prussian blue staining positive cells and fluorescent cells were located in the injury myocardium.Conclusion: This study suggests that intracoronary injection of dual labeled MSCs is probably safe and effective after myocardial infarction.
Part IV In vivo magnetic resonance imaging tracking of mesenchymal stem cells in ischemic swine heart
Objective: To track in vivo intracoronary injection of MSCs labeled with SPIO by using magnetic resonance imaging (MRI)in swine myocardial infarction model.Methods: Swine MSCs were labeled with SPIO, Cell labeling efficiency was assessed by Prussian blue stain. SPIO labeled cells underwent MRI in vitro. MR examinations were performed at 1、3and 5 week after intracoronary transplantation. Double echo steady state was used to scan four-chamber and cor biloculare at long axis view, which was considered as locating phase to obtain image of left ventricle at short axis view .The swine were euthanized at 5 week after MR examinations. Hearts were excised and sliced from apex to base into 6～8 transverse sections according to MR findings. MR findings were confirmed by histological examinations results. The transplantated labelled MSCs was tested through Prussian blue staining and fluorescent imaging.Result: The SPIO labelled MSCs transplantation intracoronary produced a hypointense signal using T2-weighted MRI and hypointense signal persisted for up to 5 weeks. Histologic analyses confirmed the presence of SPIO labelled MSCs high retention and mainly localized in the injured myocardium.Conclusion: This study demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging; MR findings consistency to pathohistological results.
Part V Sorted the fluorescently labeled transplantated cells from injury myocardium using fluorescence-activated cell sorting
Objective:To assess the utility of fluorescence-activated cell sorting (FACS) for separating CM-DiI labeled MSCs from injured myocardium.Methods: Fresh single cell suspensions were generated from injured myocardium .Fresh suspensions were sorted by FACS analysis of CM-DiI labeled fluorescent cell. FACS was performed with a FACS Vantage Flow Cytometer (Beckman Coullter ALTRA). The 488-nm argon laser was used.The purification CM-DiI labeled transplantation cells sorted from injured myocardium single cell suspensions with FACS. Selective transplantation was performed using fluorescence microscope and cells cycle was detected by flow cytometry.Result: Success of MSCs transplantation intracoronary was confirmed by FACS analysis. CM-DiI labeled transplantation cells were disassociated from injured myocardium. The purification CM-DiI labeled transplantation cells sorted from injured myocardium display red-fluorescent under fluorescence microscope. The cell cycle G2+S ( % ) was 41.6%.Conclusion: FACS techniques provide a powerful approach for analyzing and purification of transplantation MSCs. The study might provide some new clues for the design of therapeutic approaches for MSCs transplantation.
骨髓间充质干细胞(Bone marrow-derived mesenchymal stem cellsMSCs)移植可以修复受损心肌细胞、促使心肌细胞再生、改善心脏功能。MSCs移植已经成为一种新的方法用于心肌细胞再生替代治疗,为临床治疗心血管疾病开辟了一个新的领域。为了明确细胞移植后,脏器功能的改善是否确由移植细胞所引起,需要知道移植细胞能否到达心肌梗塞区域,能否在心肌梗塞区域存活。而观察移植干细胞在体内迁移和转归一直是让人困扰的问题。既往研究多通过处死实验动物行组织切片检查加以证实,这种方法不利于活体对移植细胞迁移做动态观察,也无法适用于临床研究。因此,若将干细胞移植真正应用于临床治疗,必须解决在活体如何识别、追踪移植的干细胞。本研究用超顺磁性氧化铁纳米粒子(superparamagneticiron oxide,SPIO)及CM-DiI荧光双标记骨髓间充质干细胞;通过微创法建立小型猪闭胸式心肌梗塞(myocardial infarction MI)模型;用Over-The-Wire(OTW)球囊经冠脉移植SPIO及CM-DiI双标记的骨髓间充质干细胞;通过磁共振分子成像来活体示踪移植的骨髓间充质干细胞在心肌中的分布。而且本研究还根据磁共振活体示踪结果,创新性的借用分选造血干细胞及肿瘤干细胞的方法:荧光激活细胞分离技术(fluorescence-activated cell sorting,FACS)分选出移植到心肌梗塞区域的骨髓间充质干细胞,为进一部研究骨髓间充质干细胞的作用机理奠定基础。
第一部分超顺磁性氧化铁纳米离子和CM-DiI双标记骨髓间充质干细胞
目的:观察SPIO及CM-DiI对骨髓间充质干细胞的标记效果。方法:全骨髓及密度梯度离心法分离培养猪MSCs。50μg/mL的SPIO及CM-DiI标记第3代MSCs,普鲁士蓝铁染色鉴定SPIO的标记效率,电子显微镜观察细胞内SPIO的摄取;荧光显微镜下观察CM-DiI标记效率。台盼蓝排斥实验观察标记后细胞活性,四唑盐(MTT)比色实验观察标记后细胞生长情况。结果:SPIO及CM-DiI标记MSCs效率高SPIO浓度为50μg/mL,标记时间24h对MSCs的生长、增殖无影响。结论:SPIO及CM-DiI标记猪骨髓间充质干细胞效率高,对细胞生长增殖无影响。
第二部分建立猪闭胸式心肌梗塞模型
目的:用微创法建立猪闭胸式心肌梗塞模型。方法:球囊封堵位置在第一对角支远端与第二对角支近端之间,封堵时间为60min。术中心电监护持续监测心电图变化,球囊封堵结束后冠脉造影观察远端血流中断情况。造模7周后行心肌组织病理学检查。结果:造模手术的27头猪死亡3头,24头成功建立心肌梗塞模型;球囊封堵结束后冠脉造影示前降支中远端血流中断;心肌组织病理切片示梗塞区心肌坏死完全。结论:微创法建立猪闭胸式心肌梗塞模型是一种可行、实用的心梗模型制作方法。
第三部分经冠脉移植SPIO及CM-DiI双标记骨髓间充质干细胞
目的:评价心肌梗塞后经冠脉移植骨髓间充质干细胞的效果及安全性。方法:27头猪分为5组:MI移植双标记MSCs组(n=6);MI移植SPIO加PBS组(n=6);MI移植CM-DiI单标记MSCs组(n=6);MI对照组(n=6);健康猪移植双标记MSCs组(n=3)。心肌梗塞模型建立2周后每头猪经冠脉移植1×10~8个细胞。细胞移植后5周行超声心动图检查,比较MI移植双标记MSCs组及MI对照组左室射血分数及短轴缩短率。心肌组织切片行荧光显微镜观察及普鲁士蓝铁染色,观察经冠脉移植MSCs的效果。结果:实验动物均成功完成MSCs移植,细胞移植中动物无死亡、未见ST段抬高及严重心律失常。细胞移植后5周,MI移植双标记MSCs组射血分数及短轴缩短率高于MI对照组(P＜0.05)。普鲁士蓝铁染色示移植的MSCs位于心肌梗塞区域,呈蓝色。心肌组织冰冻切片荧光显微镜观察移植的MSCs发出红色荧光,呈条带状分布。结论:经冠脉移植SPIO及CM-DiI双标记骨髓间充质干细胞是安全、有效。
第四部分磁共振活体示踪心肌内移植的骨髓间充质干细胞
目的:观察磁共振能否活体示踪到经冠脉移植的骨髓间充质干细胞。方法:SPIO标记猪MSCs,普鲁士蓝铁染色鉴定SPIO标记效率。SPIO标记的MSCs在体外行细胞水平磁共振成像。经冠脉移植骨髓间充质干细胞第1、3、5周后行猪心脏磁共振检查。采用快速梯度回波序列完成长轴位四腔心和二腔心扫描,在以长轴位四腔心和二腔心为定位相,垂直于室间隔获得左心室短轴位图像。根据磁共振活体示踪结果,从心尖向心底把心脏切成6～8片。心肌组织切片行普鲁士蓝铁染色及荧光显微镜观察,验证磁共振活体示踪结果。结果:经冠脉移植SPIO及CM-DiI标记的骨髓间充质干细胞在磁共振成像上显影,表现为低信号影,随着时间推移,低信号影逐渐减弱,并可持续至移植后5周。磁共振活体示踪结果与病理组织学检查结果一致。结论:证明磁共振能活体示踪到经冠脉移植的骨髓间充质干细胞。
第五部分荧光激活细胞分离技术分选出心梗区移植的骨髓间充质干细胞
目的:探讨荧光激活细胞分离技术从心肌梗塞区分选移植的MSCs可行性。方法:制备新鲜心肌组织单细胞悬液,用流式细胞仪(EPIC ALTRA,BeckmanCoulter)F/1通道分选,激发波长:488nm。分选出CM-DiI标记的移植到心肌梗塞区的骨髓间充质干细胞。荧光显微镜观察分选出的移植细胞,流式细胞仪检测分选出的移植细胞周期,并与移植前的MSCs比较。结果:荧光激活细胞分离技术(FACS)从心肌梗塞区分选出移植的骨髓间充质干细胞,进一步证明了磁共振活体示踪结果。分选出的移植细胞荧光显微镜下观察:细胞呈圆形,细胞均显红色荧光,与移植前的细胞类似。分选出的移植细胞周期测定结果示:G1期为58.3%,增殖指数G2+S期达41.6%。结论:荧光激活细胞分离技术(FACS)能从心肌组织分选出移植的MSCs,该方法为干细胞示踪研究提供新的思路。
Background
Coronary artery occlusion leads to ischemia and cell death in the heart. Cardiomyocyte death results in scar formation and left ventricular dilatation ultimately leads to progressive heart failure.
Bone mesenchymal stem cells(MSCs) transplantation provides a potential regenerative therapy for the heart damaged by myocardial infarction. The mechanism of MSCs transplantation therapy remains unclear. Numerous studies have been undertaken in animals and humans to analyze the efficacy of this new approach. The success of cell therapy will depend on the ability to monitor the fate of transplanted cells in vivo. However, rare studies evaluated of survival, migration, and differentiation status of transplantation MSCs. Many methods have been developed to track the destination of the injected stem cells. These tracking methods only confirm the presence of transplantion stem cells in postmortem recipient tissues .
To better understand the mechanism of cell therapy and potential beneficial effects observed in clinical trials. There is a need to track the transplanted stem cells in vivo.
Recent advances in the field of MR contrast media and cell labeling supports molecular and cellular imaging. One such approach involves the use of superparamagnetic iron oxide (SPIO) particles as a contrast agent for cell tracking. This exciting new area offers the potential for non-invasive tracking of implanted cells, ideal for monitoring in the clinical setting. The present study including: superparamagnetic iron oxide (SPIO) particles and fluorescent CM-DiI dye dual labeled MSCs;a minimal-invasive model of myocardial infarction in swine; intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarct model; in vivo magnetic resonance imaging tracking of bone marrow-derived mesenchymal stem cells via intracoronary administration; according to MR findings, sorted the fluorescently labeled transplantation cells from injury myocardium using fluorescence-activated cell sorting (FACS), a potential novel technique for tracking of transplantation cells.
Part I Superparamagnetic iron oxide particles and fluorescent CM-DiI dye dual labeled MSCs
Objective: To investigate the effects of labeling MSCs with SPIO and CM-DiI.Methods: Porcine MSCs were isolated and cultured by the whole bone marrow method and by Percoll density gradient centrifugation. MSCs were labeled with 50μg/mL SPIO and fluorescent CM-DiI dye. The labeling efficiency was tested through Prussian blue staining and fluorescent imaging.The intracellular iron uptake was also assessed with electron microscopy. Labeled MSCs viability and proliferation were determined using Trypan blue rejection method and MTT. Result: Nearly 100% of the MSCs were labeled with SPIO and CM-DiI . SPIO at 50μg/mL doses and treatment times of 24 h did not statistically affect the viability and proliferation of MSCs.Conclusion: Porcine MSCs could be efficiently and safely labeled with SPIO and CM-DiI.
Part II A minimal-invasive model of myocardial infarction in swine
Objective: To develop a closed-chest, minimal-invasive swine model of myocardial infarction.Methods: A balloon catheter was advanced into the left descending coronary artery directly beyond the second diagonal branch. The balloon was inflated and occlusion of the vessel, Angiography confirmed while ECG was continuously monitored. The balloon was deflated after 60min.Result: Myocardial infarction was successfully induced in 24 animals. After balloon occlusion, coronary angiography shows blood flow interruption in the distal left anterior descending branch.Post-mortem histological analysis revealed myocardial necrosis.Conclusion:The closed chest,minimally invasive methods represent a useful alternative for studies of myocardial infarction.
Part III Intracoronary injection of SPIO and CM-DiI dual labeled MSCs in a porcine infarc model
Objective: To evaluate the effects and safety of intracoronary infusion of mesenchymal stem cells after myocardial infarction.Methods: A total of 27 swine were divided into five groups:healthy animals with dual-labeled MSCs transplantation group(n=3);MI control group(n=6);MI with dual-labeled MSCs transplantation group(n=6); MI with SPIO and PBS transplantation group(n=6); MI with CM-DiI labeled MSCs group(n=6). 1×10~8 MSCs were delivered for one model by intracoronary injection 2 week post myocardial infarction. Heart function were observed 5 weeks after transplantation.The transplantation efficiency was tested through Prussian blue staining and fluorescent imaging.Result: During intracoronary injection of dual labeled MSCs, no adverse events were noted. Prussian blue staining positive cells and fluorescent cells were located in the injury myocardium.Conclusion: This study suggests that intracoronary injection of dual labeled MSCs is probably safe and effective after myocardial infarction.
Part IV In vivo magnetic resonance imaging tracking of mesenchymal stem cells in ischemic swine heart
Objective: To track in vivo intracoronary injection of MSCs labeled with SPIO by using magnetic resonance imaging (MRI)in swine myocardial infarction model.Methods: Swine MSCs were labeled with SPIO, Cell labeling efficiency was assessed by Prussian blue stain. SPIO labeled cells underwent MRI in vitro. MR examinations were performed at 1、3and 5 week after intracoronary transplantation. Double echo steady state was used to scan four-chamber and cor biloculare at long axis view, which was considered as locating phase to obtain image of left ventricle at short axis view .The swine were euthanized at 5 week after MR examinations. Hearts were excised and sliced from apex to base into 6～8 transverse sections according to MR findings. MR findings were confirmed by histological examinations results. The transplantated labelled MSCs was tested through Prussian blue staining and fluorescent imaging.Result: The SPIO labelled MSCs transplantation intracoronary produced a hypointense signal using T2-weighted MRI and hypointense signal persisted for up to 5 weeks. Histologic analyses confirmed the presence of SPIO labelled MSCs high retention and mainly localized in the injured myocardium.Conclusion: This study demonstrated that labeled MSCs can be reliably detected and tracked in vivo using MR imaging; MR findings consistency to pathohistological results.
Part V Sorted the fluorescently labeled transplantated cells from injury myocardium using fluorescence-activated cell sorting
Objective:To assess the utility of fluorescence-activated cell sorting (FACS) for separating CM-DiI labeled MSCs from injured myocardium.Methods: Fresh single cell suspensions were generated from injured myocardium .Fresh suspensions were sorted by FACS analysis of CM-DiI labeled fluorescent cell. FACS was performed with a FACS Vantage Flow Cytometer (Beckman Coullter ALTRA). The 488-nm argon laser was used.The purification CM-DiI labeled transplantation cells sorted from injured myocardium single cell suspensions with FACS. Selective transplantation was performed using fluorescence microscope and cells cycle was detected by flow cytometry.Result: Success of MSCs transplantation intracoronary was confirmed by FACS analysis. CM-DiI labeled transplantation cells were disassociated from injured myocardium. The purification CM-DiI labeled transplantation cells sorted from injured myocardium display red-fluorescent under fluorescence microscope. The cell cycle G2+S ( % ) was 41.6%.Conclusion: FACS techniques provide a powerful approach for analyzing and purification of transplantation MSCs. The study might provide some new clues for the design of therapeutic approaches for MSCs transplantation.
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