骨髓单个核细胞磁探针标记和自体移植后磁共振成像(英文)

Magnetic particle labeling of bone marrow-derived mononuclear cells and magnetic resonance imaging after autologous transplantation

背景:在多种移植细胞中,成体自身骨髓单个核细胞以其取材简便、无排异反应、无伦理学争议等优势成为具有临床应用价值的细胞来源。干细胞移植后,如何从受体辨别供体细胞,并观察其在受体中的生存状况,一直是让人困扰的问题。目的:应用超顺磁性氧化铁颗粒标记小型家猪骨髓单个核细胞,观察磁共振成像示踪磁探针标记细胞的可行性。设计:对照观察实验。单位:东南大学附属中大医院心血管病研究所。材料:实验于2006-04/2006-08在东南大学心血管病研究所完成。健康中国小型雄性家猪,三四月龄,体质量20～30kg,由东南大学实验动物中心提供(SYXK(苏)2002-0012)。方法:分离培养小型家猪自体骨髓单个核细胞,采用超顺磁氧化铁颗粒对骨髓单个核细胞分离当日细胞悬液中进行标记,普鲁士蓝染色显示细胞内铁,台盼蓝拒染法评估细胞活力。将11只用于心肌梗死模型制备的猪分为实验组9只和对照组2只。选择经皮行左或右颈动脉或股动脉穿刺途径,以1.5～2.0mm球囊封堵左前降支下1/3,304～405kPa,持续封堵60min后,术前反复予缺血预适应三四次。由猪体表心电图证实心肌梗死模型成功后,经皮冠脉内骨髓单个核细胞输注:梗死模型建立24h后,经皮股动脉穿刺行冠脉造影,应用OTW导管冠脉内输注骨髓单个核细胞细胞悬液,然后将盛放细胞的注射器及OTW导管以肝素生理盐水冲洗注入残余细胞,注射时间10min,对照组2只猪经同样方法注入未标记细胞。术后磁共振活体示踪骨髓单个核细胞并与心肌组织切片普鲁士蓝染色对照。结果:实验组7只和对照组1只小型家猪进入结果分析,两组各1只死于心肌梗死模型制备,实验组1只死于磁共振检查前麻醉意外。①骨髓单个核细胞的超顺磁氧化铁颗粒标记率近100%,在含Fe2O3-多聚左旋赖氨酸的培养基中骨髓单个核细胞可继续增殖,细胞形态无明显变化。②8只心肌梗死模型中,5只标记细胞移植后T2*WI序列心肌梗死周边见模糊的低信号区,4周后低回声信号均消失,离体T2*WI序列示梗死区周边一个呈点状分布的低信号区。③组织学检查发现5只(细胞数106以上)普鲁士蓝细胞阳性,分布部位与磁共振信号减低区一致。结论:超顺磁氧化铁颗粒标记骨髓单个核细胞安全、有效;T2*WI对磁标记骨髓单个核细胞的示踪最敏感;磁共振可对经冠脉途径植入的磁标记的干细胞进行活体示踪,磁共振信号改变与干细胞数目及分裂增殖状态相关。

BACKGROUND： Among many transplanted cells, adult autologous bone barrow-derived mononuclear cells have been used in clinical practice because they are easy to be obtained, without immunological rejection and ethical disputation and other advantages. How to distinguish donor cells from receptors and observe the survival of donor cells following stem cell transplantation still trouble people. OBJECTIVE： Superparamagnetic iron oxide （SPIO） particles-labeled bone marrow-derived mononuclear cells from minipigs were used to observe the feasibility of in vivo tracking with magnetic resonance imaging （MRI）. DESIGN ： A controlled observation experiment. SETTING： Institute of Cardiovascular Disease, Zhongda Hospital Affiliated to Southeast University MATERIALS： This experiment was carried out in the Institute of Cardiovascular Disease, Zhongda Hospital Affiliated to Southeast University between April 2006 and August 2006. Healthy Chinese minipigs, aged 3 to 4 months, weighing from 20 to 30 kg, were provided by the Experimental Animal Center of Southeast University [SYXK（Su ）2002-0012]. METHODS： Autologous bone marrow-derived mononuclear cells of minipigs were isolated and cultured. Bone marrow-derived mononuclear cells in the suspension were traced with SPIO particles. Ferrum in the cells were shown by Prussian blue staining, and cell viability was evaluated by trypan blue exclusion method. Eleven minipigs used for preparation of model of myocardial infarction were divided into experimental group （n =9） and control group （n =2）. By means of percutaneous left or right cervical artery or femoral artery puncturation, 1.5 to 2.0 mm balloon was used to occlude 1/3 left anterior descending branch, 304 to 405 kPa, 60 minutes later, ischemic preconditioning was conducted 3 to 4 times before operation. When pig models of myocardial infarction were successful that was proved by surface electrocardiogram, bone marrow-derived mononuclear cells were percutaneously injected into coronary artery. Coronary arteriography was performed through femoral artery acupuncture at 24 hours after establishing infarction models. Suspension of bone marrow-derived mononuclear cells was perfused into coronary artery with OTW catheter. Then, the injector and OTW catheter for containing cells were rinsed with normal saline containing heparin and infused with the residual cells within 10 minutes. Non-labeled cells were perfused in 2 minipigs of control group by the same method. Postoperatively, bone marrow-derived mononuclear cells were traced by magnetic resonance and compared with Prussian blue-stained myocardial tissue sections. RESULTS： Seven minipigs of experimental group and one minipig of control group were involved in the final analysis. One of each group was used for preparation of model of myocardial infarction. One minipig of experimental group died from anesthetic accident before magnetic resonance. （1） Bone marrow-derived mononuclear cells all were nearly labeled by SPIO particles. Bone marrow-derived mononuclear cells could further proliferate in culture medium containing Fe203-PLL without obvious changes of cellular shape. （2）T2^＊ Wl showed that 5 of 8 models of myocardial infarction presented fuzzy low-echo signal region in peripheral myocardial infarction after transplantation of labeled cells and the low-echo signal disappeared 4 weeks later. Ex vivo T2^＊ Wl sequence showed there was a dot-distributed low-echo signal region in the peripheral infarction region. （3） It was found in histological examination that 5 models （ cell number over 10^6） had Prussian blue-positive cells, which distributed the same as those in magnetic resonance signal reducing region. CONCLUSION ： SPIO particles-labeled bone marrow-derived mononuclear cells are safe and effective; T2^＊ Wl is sensitive to tracing SPIO particles-labeled bone marrow-derived mononuclear cells; Magnetic resonance can in vivo trace SPIO particles-labeled stem cells transplanted through coronary artery, magnetic resonance signal change is related with the number of stem cells and division growth.