【作者】 宋鹏；

【导师】 胡盛寿；

【作者基本信息】 中国协和医科大学 ， 心血管外科， 2008， 博士

【摘要】 第一部分:不同心脏状态下心梗边缘区注射骨髓间充质干细胞的比较目的:细胞移植是目前缺血性心脏病治疗研究的热点之一。干细胞治疗心肌梗塞已被证明是种很有前途的治疗方法。细胞移植在体内的存留、分布、迁移除了受移植途径影响外,心脏的状态也会影响移植细胞的归宿。本实验主要研究停跳与不停跳两种心脏状态下,经猪心梗模型心肌注射骨髓间充质干细胞后全身细胞的分布情况。方法:实验方法分为体内和体外实验两部分体外实验部分:从雄性中华小型猪髂骨处抽取骨髓,体外培养扩增骨髓间充质干细胞并进行鉴定。将超顺磁氧化铁颗粒和骨髓间充质干细胞共同孵育培养36-48小时。使用普鲁士蓝染色评价标记后的细胞铁离子标记的效率;使用台盼蓝染色检验标记后细胞的活性。体内实验部分:用带球囊血管造影导管经股动脉导入雌性猪左冠状动脉前降支,在第二对角支的近端将球囊扩张90分钟造成急性心梗模型。模型建立后7天随机分为四组。第1组为停跳心脏细胞注射组(n=6),体外循环后心脏停跳,标记的细胞(1×10~8)经心肌注入心梗周边区。第2组为不停跳心脏细胞注射组(n=6),相同的细胞在跳动下心脏经心肌注入心梗周边区。第3组停跳心脏对照组(n=6)和第4组不停跳心脏对照组(n=6)中,相同剂量的生理盐水分别在停跳和不停跳心脏状态下经心肌注入心梗周边区。3天后,体内细胞分布通过核磁共振的T2*变化及雄性细胞特异性的SRY基因的实时定量PCR检测来评价。并取动物心,肝,脾,肺,肾的标本,切片后进行普鲁士蓝染色,观察细胞在体内的分布和形态。结果体外实验部分:普鲁士兰染色的超顺磁铁纳米颗粒标记的骨髓间充质干细胞显示,蓝染的铁的小囊泡或小颗粒分布于几乎每个细胞的胞浆中,其标记效率几乎达到100%。并且,在台盼兰排斥实验中,95%以上的细胞拒染台盼兰,可见骨髓间充质干细胞经铁标记后有良好的活性。体内实验部分:细胞可以在心,脾,肺和肝中检测到。1,2组中大部分的注射细胞滞留在心脏,并目1组中心脏细胞的滞留较2组多(T2*改变:22.3±2.2 versus 17.00±0.84;SRY gene:0.150±0.062 versus 0.072±0.003)。病理学检查可以见移植细胞呈普鲁士蓝染色阳性,并与磁共振影像学的显影有很好的一致性。结论:经心肌注射骨髓间充质干细胞后,许多细胞流向心脏以外的器官,尤其是脾脏。在心脏停跳状态下经心肌注射骨髓间充质干细胞将更有利于细胞在心脏的滞留,在开胸手术中是一种最适合的细胞移植方法。第二部分:体外循环和停跳的心脏状态对于经冠状动脉内移植骨髓间充质细胞后细胞的存留、分布的影响目的:冠状动脉内细胞移植是目前临床细胞移植的主要途径,冠状动脉内细胞移植后移植细胞的存留、分布和迁移是关系细胞移植治疗效果的重要因素之一。体外循环和停跳的心脏状态是心脏外科手术常用的方式,本实验主要比较停跳与不停跳两种心脏状态下经冠脉注射骨髓间充质干细胞后移植细胞在心脏内的存留、分布和迁移情况以及在全身重要脏器内的分布。方法:雄性猪的骨髓间充质干细胞用超顺磁铁纳米颗粒标记。雌性猪心梗后一周随机分为四组。第1组为跳动心脏细胞注射组(n=6),标记的细胞(1×10~8)经左前降支冠脉注入跳动的心脏。第2组为停跳心脏细胞注射组(n=6),体外循环建立,心脏停跳后,相同的细胞经左前降支注入。第3组跳动心脏对照组(n=6)和第4组停跳心脏对照组(n=6)中,相同剂量的生理盐水分别在不停跳和停跳心脏状态下经冠脉注入心脏。3天后,体内细胞分布通过核磁共振的T2*变化及Y染色体性别决定区基因(SRY基因)的实时定量PCR检测来评价。结果:细胞可以在心,肝,脾,肺和肾中检测到。1,2组中一小部分的注射细胞滞留在心脏,但两组比较没有统计学差异。大量的细胞滞留在心脏外脏器中,尤以停跳细胞移植组脾脏中较多。1组中移植细胞的凋亡比率较2组少,在细胞移植后3天未发现有肌样分化表现。结论:经冠脉移植细胞后大量的细胞将滞留在心外器官脾脏中,停跳心脏对于冠脉移植干细胞并无明显意义。最适宜的间充质干细胞的移植方式仍需进一步探索。更多还原

【Abstract】 Objective:Stem cell therapy has emerged as a promising approach for the treatment of myocardial infarction(MI).The transplanted cell’s retention, migration is affected not only by the transplanted approach,but also by the heart status.This preclinical study was to test the intramyocardially injected bone marrow(BM) mesenchymal stem cells(MSCs) cell distribution in different heart statues(arresting or beating) in a porcine myocardial infarction (MI) model.Methods:In vitro study:BM was aspirated from the iliac crest of male swine.Bone marrow MSCs were expanded and incubated with SPIO for 48 hours.The labeling efficiency was tested through Prussian blue staining,and cells viability was tested through Trypan blue rejection method.In vivo study:An occlusive angioplasty balloon was advanced into the proximal left anterior descending coronary artery via percutaneous approach.Acute MI was induced by inflating the balloon for 90 minutes followed by artery perfusion.1 week after creation of MI in female swine,the survivals were randomly divided into 4 groups.Cardiopulmonary bypass was set up to arrest heart,and then labeled cells(1×10~8) were intramyocardially injected into the borderline of infracted zone in Group 1(n=6).Same volumes of cells were grafted in the beating heart in Group 2(n=6).In Group 3 and 4,the same volume of saline was injected in either arresting or beating heart(n=6 respectively).3 days later,cell distribution was assessed by T2~* change with magnetic resonance imaging and sex-determining region on Y-chromosome (SRY) with quantitive polymerase chain reaction.The interested segment of heart,liver,spleen,lung,and kidney tissues were collected for Prussian Blue Staining.Results:In vitro study:Prussian blue staining of SPIO-labeled MSCs revealed the presence of numerous blue staining iron-containing vesicles or particles in the cytoplasm of nearly every cell.The labeling efficiency reached approximately 100%.More than 95%labeled cells were not stained with Trypan Blue.In vivo study:The cells were identified in heart,spleen,lung and liver.Most of injected cells were localized in the myocardium in Group 1 and 2,however, the amount of detained cells was much higher in Group 1(T2~* change: 22.3±2.2 versus 17.00±0.84;SRY gene:0.150±0.062 versus 0.072±0.003). The injected sites containing labeled cells could all be detected through MRI and were confirmed on pathology.Conclusion:Even after intramyocardially injection,many cells migrated to extracardiac organs especially to the spleen.Our results indicated injection in the arresting heart could favor more cells be retained in myocardium and thus it was an optimal approach to deliver MSCs during open chest surgery. Backgroud:Transcorocnary infusion of the therapeutic stem cells is the most popular delivery approach of cell transplantation to improve cardiac function for the injured heart,but few data is reported about the retention,distribution, and migration of the implanted cells.Systemic distribution of bone marrow stromal cells(BMSCs) after intracoronary infusion(ICI) and the role of cardiopulmonary bypass(CPB) in cell distribution still remain unclear.This study was designed o analyze the cell distribution after ICI in variations of heart status in a swine myocardial infarction(MI) model.Methods:After inducing an MI,iron oxide labeled male cells(1×10~8) were infused through the coronary artery of the beating swine heart in Group 1.In group 2,CPB was set up and then the same volume of cells was infused after cadioplegic arrest.In Group 3 and 4,the animals underwent either beating or arrested ICI with the same volume of saline.Three days later,cell distribution was assessed by T2~* change with magnetic resonance imaging and sex-determining region on Y-chromosome with quantitive polymerase chain reaction. Results:Only a few transplanted cells were localized in the heart and no difference was found between groups 1 and 2.The majority of BMSCs would be trapped in extracardial organs,and more cells resided in the spleen in arrested heart status.The percentage of the apoptotic cells in the implanted cells in Group 1 was lower than that in Group 2.Expression of the muscle-specific markers wasn’t observed at 3 days in Prussian blue-positive cells.Conclusion:The majority of BMSCs transplanted by ICI would be entrapped by the extracardial organs.The arrested heart with CPB during ICI does not favor more cells retention in the injured myocardium.The optimal approach of delivery of BMSCs still needs further investigation.更多还原