光学—磁共振双模监测间充质干细胞治疗急性心肌梗死的实验研究

发布时间：2018-09-07 21:32

【摘要】：目的：采用萤火虫荧光素酶(Fluc)和荧光染料CyI (Cy5的衍生物)标记SD大鼠骨髓间充质干细胞(BMSCs),体外研究光学标记干细胞的有效性。将CyI染料标记的BMSCs/Fluc植入不同体重大鼠、小鼠急性心梗模型心肌组织中,探索在不同动物模型中光学示踪干细胞的可行性。结合超小超顺磁性氧化铁颗粒(USPIO)标记BMSCs/Fluc,双模态示踪干细胞在植入心肌组织后的存活、增殖、迁移和畸胎瘤形成。方法：将成功转染、稳定表达Fluc的SD大鼠BMSCs/Fluc配制成不同浓度细胞液,行体外生物发光成像(BLI),验证Fluc标记有效性。采用不同CyI浓度和孵育时间对BMSCs/Fluc进行孵育。行体外荧光成像,确定恰当孵育条件。使用含USPIO(40μg/ml)和多聚赖氨酸(PLL,1.5μg/ml)的培养基与BMSCs/Fluc共孵育24h,普鲁士蓝染色观察铁颗粒细胞内分布。200g、150g、100gSD大鼠、20-25g Bal b/c小鼠各5只,开胸结扎冠脉前降支构建心梗模型,心肌植入CyI标记BMSCs/Fluc,术后第1天行BLI,选择阳性动物模型(Bal b/c小鼠)行后续实验。Bal b/c小鼠心梗模型心肌植入USPIO标记BMSCs/Fluc,术后第1、3、6、9天行生物发光成像,术后第3天行磁共振检查。取病理行HE染色和普鲁士蓝染色观察心肌组织USPIO颗粒分布情况。结果：CyI孵育浓度1.0×10-6、孵育时间4h时,荧光强度最强,死细胞数未见明显变化。CyI与Fluc光信号强度均随细胞数增加而线性增加。在体光学成像中,不同体重大鼠及小鼠心梗模型心脏均未见CyI荧光信号,离体可见。Bal b/c小鼠心梗模型BLI可见光信号,但SD大鼠BLI未见。USPIO标记BMSCs/Fluc植入Bal b/c小鼠心梗模型心肌组织中后,BLI光强度逐渐减少,9天内降至0,表明干细胞逐渐死亡,未见增殖、迁移及畸胎瘤形成。磁共振可见左心室前壁低信号区域。病理结果显示USPIO与移植干细胞在心肌的分布一致。结论： 1、CyI孵育浓度1.0×10-6mol/L孵育时间4h可安全有效标记BMSCs/Fluc,且体外检测荧光强度随细胞数量增加而增加,呈线性关系； 2、Fluc可对Bal b/c小鼠心梗模型心肌局部注射干细胞进行在体生物发光成像,CyI荧光染料标记可在离体条件下示踪移植干细胞； 3、USPIO标记的BMSCs/Fluc植入Bal b/c小鼠心梗模型心肌组织中后,生物发光信号逐渐减少,表明干细胞逐渐死亡,未见增殖、迁移及畸胎瘤形成；MRI可对心肌移植的USPIO标记的BMSCs/Fluc进行在体示踪；病理结果显示USPIO与移植干细胞在心肌的分布一致
[Abstract]:Aim: to investigate the effectiveness of optical labeling of SD rat bone marrow mesenchymal stem cells (BMSCs) with fluorescent luciferase (Fluc) and fluorescent dye CyI (Cy5 derivative) in vitro. BMSCs/Fluc labeled with CyI dye was implanted into myocardial tissue of rats with different body weight and acute myocardial infarction in mice to explore the feasibility of optical tracing stem cells in different animal models. The survival, proliferation, migration and teratoma formation of ultrasmall superparamagnetic iron oxide (USPIO) labeled BMSCs/Fluc, double mode tracer stem cells after implantation of myocardial tissue. Methods: SD rat BMSCs/Fluc which was successfully transfected and stably expressed Fluc was prepared into different concentrations of cell fluid. In vitro bioluminescence imaging (BLI),) was performed to verify the effectiveness of Fluc labeling. BMSCs/Fluc was incubated with different CyI concentration and incubation time. Fluorescence imaging in vitro was performed to determine the appropriate incubation conditions. The culture medium containing USPIO (40 渭 g/ml) and polylysine (PLL,1.5 渭 g/ml) was incubated with BMSCs/Fluc for 24 h. Prussian blue staining was used to observe the distribution of iron granulosa cells in 5 rats with 20 to 25 g Bal b / c each. The myocardial infarction model was established by ligation of the anterior descending coronary artery. The BLI, selective positive animal model (Bal b / c mice) was performed on the first day after the implantation of CyI labeled BMSCs/Fluc, (Bal b / c mice). The myocardial infarction model of Bal / b / c mice was implanted with USPIO labeled with USPIO for 6 days, then bioluminescent imaging was performed on the first day, and magnetic resonance imaging was performed on the third day after the operation. HE staining and Prussian blue staining were used to observe the distribution of USPIO particles in myocardium. Results the fluorescence intensity was the strongest at the concentration of 1.0 脳 10 ~ (-6) and the incubation time of 4 h. The number of dead cells did not change obviously. The intensity of light signal of both Cyi and Fluc increased linearly with the increase of the number of cells. In vivo optical imaging, there was no CyI fluorescence signal in heart of rats and mice with different body weight, but BLI visible signal was observed in isolated myocardial infarction model of Bal / b / c mice. However, BLI of SD rats did not see. USPIO-labeled BMSCs/Fluc was implanted into myocardial tissue of Bal b / c mice. The light intensity of BLI decreased gradually within 9 days, indicating that stem cells died gradually, no proliferation, migration and teratoma formation. Mr imaging showed the left ventricular anterior wall with low signal intensity. Pathological results showed that the distribution of USPIO was consistent with that of transplanted stem cells in myocardium. Conclusion: (1) the concentration of Cyi was 1.0 脳 10-6mol/L for 4 h, and the fluorescence intensity increased with the increase of the number of cells in vitro, which showed a linear relationship. 2Fluc could locally inject stem cells into myocardium of Bal b / c mouse myocardial infarction model. In vivo bioluminescent imaging with Cyi fluorescent dye could be used to trace the stem cells to transplanted stem cells in vitro. 3After implantation of BMSCs/Fluc labeled with USPIO into myocardial tissue of Bal b / c mice, the bioluminescence signal decreased gradually, indicating that stem cells died gradually, without proliferation, migration and teratoma formation. MRI can trace the BMSCs/Fluc labeled by USPIO in vivo and the pathological results show that the distribution of USPIO and transplanted stem cells in myocardium is the same as that of transplanted stem cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R542.22

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