MRI对急性心肌梗死后不同心脏状态下干细胞移植后体内再分布与疗效评价的动物实验研究

目的 探讨MRI在研究停跳与不停跳两种心脏状态下,经猪心肌梗死模型心肌注射骨髓间充质干细胞后全身各主要脏器(心、肝、脾、肾)干细胞的早期再分布及疗效评价中的应用价值.方法 干细胞提取自雄性猪并用超顺磁性氧化铁标记.雌性猪制成急性心肌梗死模型7 d后数字表随机方法分为4组.第1组为心脏停跳细胞注射组(6只),体外循环后心脏停跳,超顺磁性氧化铁标记的干细胞(1×108)经心肌注入心肌梗死周边区.第2组为不停跳心脏细胞注射组(6只),相同的细胞在心脏跳动下经心肌注入心肌梗死周边区.第3组停跳心脏对照组(6只)和第4组不停跳心脏对照组(6只)中,相同剂量的生理盐水分别在停跳和不停跳状态下经心肌注入心肌梗死周边区.3 d后,行MR T2*WI示踪和心功能检查.并取动物心、肝、脾、肺、肾的标本,用实时定量聚合酶链反应(qRT-PCR)检测雄性细胞特异性的性别决定区域(SRY)基因定量检查.统计学分析采用方差分析和t检验.结果 第1～4组术前左心室舒张末期容积(LVEDV)分别为(56.8±5.3)、(54.8±6.8)、(57.4±4.3)和(56.8±2.8) ml,术后LVEDV分别为(65.2±5.2)、(63.2±3.7)、(60.2±4.7)和(62.2±4.4) ml,术前左心室收缩末期容积(LVESV)分别为(33.5±7.6)、(32.3±5.3)、(33.5±3.6)和(32.7±4.6) ml,术后LVESV分别为(37.3±5.6)、(36.3±6.9)、(34.3±5.4)和(36.3±8.1) ml;术前左心室射血分数值(LVEF)分别为(42.3±7.2)%、(41.7±6.8)%、(41.8±8.6)%和(42.7±7.7)%,术后LVEF分别为(44.5±8.7)%、(43.1±7.4)%、(42.8±5.6)%和(43.3±8.4)%;术前心肌梗死面积(MI)分别为(6.5±2.1)、(6.4±1.9)、(6.5±2.5)和(6.4±2.6) cm2,术后MI分别为(6.4±2.3)、(6.2±2.6)、(6.3±2.5)和(6.4±2.8) cm2,差异均无统计学意义(术前P值均＞0.05,F值分别为0.277、0.066、0.066、0.003;术后P值均＞0.05,F值分别为1.137、0.182、0.021及0.008).心脏中第1组较第2组T2*降低显著(-22.3±2.2)和(-17.0±0.8) ms,t=-5.489,P＜0.01],而脾脏中第2组较第1组T2*值降低显著(-7.7±0.7)和(-13.3±1.1) ms,t=9.055,P＜0.01],在肝脏及肾脏2组差异无统计学意义(肝脏t=-0.532,P>0.05,肾脏t=-0.113,P>0.05).SRY基因和qRT-PCR结果,第1组及第2组心脏(150±62)和(72±4) U/L]、脾脏(131±1)和(233±17) U/L]及肝脏(17±1)和(9±5) U/L]差异有统计学意义(P值均＜0.05,t值分别为3.109、-13.286及3.492),病理学检查可以见移植细胞呈普鲁士蓝染色阳性,与MRI有很好的一致性.结论 干细胞氧化铁颗粒标记后,MRI可以作为方便而有效的手段在移植早期活体示踪干细胞,评价其在体内的分布情况.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.

Abstract：

Objective To evaluate the efficacy of MRI for assessment of re-distribution of bone marrow mesenchymal stem cells injected intramyocardially in main organs (heart, liver, spleen and kidney) under different heart status (beating or arresting) in a porcine model. Methods Bone marrow-derived mesenchymal stem cells were obtained from the male swine and labeled with iron oxide during culture. Acute myocardial infarction was created in female swine, one week later, the survivors were randomly divided into 4 groups. Cardiopulmonary bypass was set up to arrest the heart, and then labeled cells (1×108) were intramyocardially injected into the border of the infracted myocardium in group 1 (n=6). The same volume of cells was grafted into the beating heart in group 2 (n=6). In group 3 and 4, saline was injected into either the arresting or beating myocardium. Three days later, re-distribution of stem cells and cardiac function were assessed by T2*WI and cine MRI, respectively. All animals were sacrificed for histology and real-time quantitative polymerase chain reaction (RT-PCR) of sex-determining region on Y-chromosome (SRY) investigation.The ANOVA and t test was used for statistics. Results The left ventricular end-diastolic volume (LVEDV) before transplantation for group 1-4 were: (56.8±5.3),(54.8±6.8),(57.4±4.3)and(56.8±2.8) ml, and after transplantation for group 1-4 were: (65.2±5.2),(63.2±3.7),(60.2±4.7)and(62.2±4.4) ml. The left ventricular end-systolic volume (LVESV) before transplantation for group 1-4 were: (33.5±7.6),(32.3±5.3),(33.5±3.6)and(32.7±4.6) ml,and after transplantation for group 1-4 were: (37.3±5.6),(36.3±6.9),(34.3±5.4)and(36.3±8.1) ml. The left ventricular EF values (LVEF) before transplantation for group 1-4 were: (42.3±7.2)%,(41.7±6.8)%,(41.8±8.6)% and(42.7±7.7)%,and after transplantation for group 1-4 were: (44.5±8.7)%,(43.1±7.4)%,(42.8±5.6)% and(43.3±8.4)%. The myocardial infarction area (MI) before transplantation for group 1-4 were: (6.5±2.1),(6.4±1.9),(6.5±2.5)and(6.4±2.6) cm2,and after transplantation for group 1-4 were: (6.4±2.3),(6.2±2.6),(6.3±2.5)and(6.4±2.8) cm2 . There were no statistical differences before and after transplantation in these 4 groupsP values of before and after transplantation for LVEDV, LVESV, LVEF,MI were >0.05 (F= 0.277, 0.066,0.066, 0.003); and >0.05 (F= 1.137,0.182,0.021,0.008),respectively]. The T2 value of the infracted myocardium in group 1 decreased more obviously than that in group 2(-22.3 ± 2.2) vs (-17.0 ± 0.8) ms, t=-5.489, P＜0.01], while the T2 value of the spleen decreased more significantly in group 2 than that in group 1(-7.7 ± 0.7) vs (-13.3 ± 1.1) ms,t=9.055, P＜0.01]. The T2 values of the liver and kidney were no significant differences in group 1 and 2 (liver, t=-0.532,P>0.05 and kidney, t=-0.113,P>0.05). The results of RT-PCR in group 1 and 2 showed significant differences in heart(150±62) vs (72±4) U/L ,P<0.05, t=3.109], spleen(131±1) vs (233±17) U/L, P＜0.01, t=- 13.286]and liver(17±1) vs (9±5) U/L ,P<0.01,t= 3.492]. Pathological examination demonstrated that the transplanted stem cells were positive for Prussian blue staining, which had a good correlation with MRI results. Conclusion MRI can serve as a convenient and efficient imaging method to track the migration of stem cells with SPIO labeled in early stage and evaluate its early re-distribution in vivo. Injection of bone marrow mesenchymal stem cells in the arresting heart could favor retaining more cells in the myocardium.

Assessment of re-distribution and efficacy of stem cell transplantation in different heart status after acute myocardial infarction by MRI: an experimental study

Objective To evaluate the efficacy of MRI for assessment of re-distribution of bone marrow mesenchymal stem cells injected intramyocardially in main organs (heart, liver, spleen and kidney) under different heart status (beating or arresting) in a porcine model. Methods Bone marrow-derived mesenchymal stem cells were obtained from the male swine and labeled with iron oxide during culture. Acute myocardial infarction was created in female swine, one week later, the survivors were randomly divided into 4 groups. Cardiopulmonary bypass was set up to arrest the heart, and then labeled cells (1×108) were intramyocardially injected into the border of the infracted myocardium in group 1 (n=6). The same volume of cells was grafted into the beating heart in group 2 (n=6). In group 3 and 4, saline was injected into either the arresting or beating myocardium. Three days later, re-distribution of stem cells and cardiac function were assessed by T2*WI and cine MRI, respectively. All animals were sacrificed for histology and real-time quantitative polymerase chain reaction (RT-PCR) of sex-determining region on Y-chromosome (SRY) investigation.The ANOVA and t test was used for statistics. Results The left ventricular end-diastolic volume (LVEDV) before transplantation for group 1-4 were: (56.8±5.3),(54.8±6.8),(57.4±4.3)and(56.8±2.8) ml, and after transplantation for group 1-4 were: (65.2±5.2),(63.2±3.7),(60.2±4.7)and(62.2±4.4) ml. The left ventricular end-systolic volume (LVESV) before transplantation for group 1-4 were: (33.5±7.6),(32.3±5.3),(33.5±3.6)and(32.7±4.6) ml,and after transplantation for group 1-4 were: (37.3±5.6),(36.3±6.9),(34.3±5.4)and(36.3±8.1) ml. The left ventricular EF values (LVEF) before transplantation for group 1-4 were: (42.3±7.2)%,(41.7±6.8)%,(41.8±8.6)% and(42.7±7.7)%,and after transplantation for group 1-4 were: (44.5±8.7)%,(43.1±7.4)%,(42.8±5.6)% and(43.3±8.4)%. The myocardial infarction area (MI) before transplantation for group 1-4 were: (6.5±2.1),(6.4±1.9),(6.5±2.5)and(6.4±2.6) cm2,and after transplantation for group 1-4 were: (6.4±2.3),(6.2±2.6),(6.3±2.5)and(6.4±2.8) cm2 . There were no statistical differences before and after transplantation in these 4 groupsP values of before and after transplantation for LVEDV, LVESV, LVEF,MI were >0.05 (F= 0.277, 0.066,0.066, 0.003); and >0.05 (F= 1.137,0.182,0.021,0.008),respectively]. The T2 value of the infracted myocardium in group 1 decreased more obviously than that in group 2(-22.3 ± 2.2) vs (-17.0 ± 0.8) ms, t=-5.489, P＜0.01], while the T2 value of the spleen decreased more significantly in group 2 than that in group 1(-7.7 ± 0.7) vs (-13.3 ± 1.1) ms,t=9.055, P＜0.01]. The T2 values of the liver and kidney were no significant differences in group 1 and 2 (liver, t=-0.532,P>0.05 and kidney, t=-0.113,P>0.05). The results of RT-PCR in group 1 and 2 showed significant differences in heart(150±62) vs (72±4) U/L ,P<0.05, t=3.109], spleen(131±1) vs (233±17) U/L, P＜0.01, t=- 13.286]and liver(17±1) vs (9±5) U/L ,P<0.01,t= 3.492]. Pathological examination demonstrated that the transplanted stem cells were positive for Prussian blue staining, which had a good correlation with MRI results. Conclusion MRI can serve as a convenient and efficient imaging method to track the migration of stem cells with SPIO labeled in early stage and evaluate its early re-distribution in vivo. Injection of bone marrow mesenchymal stem cells in the arresting heart could favor retaining more cells in the myocardium.