双模态标记F344大鼠骨髓间充质干细胞体外及体内实验研究初探
发布时间：2019-04-20 17:32
【摘要】：目的：利用红色荧光蛋白(RFP)和荧光染料CyI (Cy5的衍生物)标记F344大鼠骨髓间充质干细胞(BMSCs),研究光学标记干细胞的安全及有效性、在大鼠体表对标记干细胞成像的可行性,并结合超小超顺磁性氧化铁颗粒(USPIO)同时标记BMSCs,探索在体双模态标记干细胞示踪成像的可能。 方法：使用成功转染、稳定表达RFP的F344大鼠BMSCs (BMSCs/RFP);同时,使用含不同浓度的CyI培养基与BMSCs/RFP共孵育培养24h后。在体外,用荧光检测仪分别检测荧光强度,用光学成像系统分别检测体表注射光学标记细胞的F344大鼠成像效果。使用含USPIO (40μg/ml)和多聚赖氨酸(PLL,1.5μg/ml)的培养基与BMSCs/RFP共孵育24h,标记后行普鲁士蓝染色观察铁颗粒在细胞内的分布。通过皮下或肌肉注射RFP或CyI标记的BMSCs,进行大鼠体表的光学成像。活体条件下,开胸结扎冠状动脉左前降支(LAD)建立F344大鼠急性心梗模型,通过心肌局部注射移植三种标记细胞(BMSCs/RFP、CyI或USPIO标记的BMSCs/RFP,其中以USPIO/RFP双标的为主),利用光学成像及MRI进行标记细胞的在体示踪。取病理行普鲁士蓝染色及荧光成像观察心脏中USPIO颗粒及RFP的分布情况。 结果：体外荧光强度检测显示RFP或CyI标记(CyI孵育细胞浓度为5.0×10-5mol/L)的BMSCs在细胞浓度达到5.0×105/ml以上时有较强的信号强度；当CyI与BMSCs/RFP共孵育时,其孵育浓度分别在5.0×10-6、1.0×10-5及5.0×104mol/L时细胞形态及死细胞数未见明显变化。大鼠体表成像时在注射细胞数为1.0×106条件下,CyI的荧光成像效果优于RFP。呼吸机辅助通气条件下通过开胸结扎冠状动脉左前降支(LAD)可成功建立F344大鼠急性心梗模型。通过心肌局部注射的标记细胞在光学条件下,在体成像没有检测到荧光信号,离体成像能检测到心脏表面有较弱的荧光：MRI在标记后2周内均能观察到注射区域信号强度明显降低。病理普鲁士蓝染色及荧光成像提示心肌内铁颗粒及RFP分布与移植干细胞分布一致。 结论： 1、RFP或CyI(CyI孵育细胞浓度为5.0×10-5mol/L)可以安全有效地标记BMSCs; 2、RFP或CyI标记的BMSCs在F344大鼠体表的光学成像是可行的； 3、离体成像可示踪心脏局部注射光学标记的BMSCs; 4、MRI在标记后2周仍可在体示踪USPIO标记的BMSCs.
[Abstract]:Objective: to study the safety and efficacy of F344 rat bone marrow mesenchymal stem cells (BMSCs) labeled with red fluorescent protein (RFP) and fluorescent dye CyI (derivatives of Cy5). The feasibility of imaging the labeled stem cells on the surface of the rat was studied, and the possibility of bimodal labeled stem cell tracer imaging in vivo was explored by combining ultra-small superparamagnetic iron oxide particles with (USPIO) and labeling BMSCs, simultaneously. Methods: F344 rat BMSCs (BMSCs/RFP) expressing RFP stably was successfully transfected and co-cultured with BMSCs/RFP with different concentration of CyI for 24 h. In vitro, the fluorescence intensity was measured by fluorescence detector, and the imaging effect of F344 rats injected with optically labeled cells on the body surface was detected by optical imaging system. The medium containing USPIO (40 渭 g / ml) and polylysine (PLL, 1.5 渭 g / ml) was incubated with BMSCs/RFP for 24 h. The distribution of iron particles in the cells was observed by Prussian blue staining. Optical imaging of rat body surface was performed by subcutaneous or intramuscular injection of RFP or CyI labeled BMSCs,. In vivo, a F344 rat model of acute myocardial infarction was established by ligating the left anterior descending branch of the coronary artery (LAD) by thoracotomy. Three kinds of labeled cells (BMSCs/RFP,CyI or USPIO labeled BMSCs/RFP, in which USPIO/RFP was double labeled) were injected into the myocardium of F344 rats. Optical imaging and MRI were used to label the cells in vivo. Prussian blue staining and fluorescence imaging were used to observe the distribution of USPIO particles and RFP in heart. Results: in vitro fluorescence intensity analysis showed that BMSCs labeled with RFP or CyI (5.0 脳 10-5mol/L in CyI) had a strong signal intensity when the cell concentration was higher than 5.0 脳 105/ml. When the concentration of CyI and BMSCs/RFP were 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L respectively, the cell morphology and the number of dead cells did not change significantly at the concentration of 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L. When the number of injected cells was 1.0 脳 10 6, the fluorescence imaging effect of CyI was better than that of RFP. in rat body surface imaging. The acute myocardial infarction model of F344 rats was successfully established by ligating the left anterior descending coronary artery (LAD) under ventilator-assisted ventilation. Labeled cells injected locally into the myocardium did not detect fluorescence signals in vivo imaging under optical conditions. In vitro imaging was able to detect weak fluorescence on the heart surface: the signal intensity of the injection region was significantly decreased by MRI within 2 weeks after labeling. Pathological Prussian blue staining and fluorescence imaging suggested that the distribution of iron particles and RFP in myocardium was consistent with that of stem cells transplantation. Conclusion: 1. The concentration of RFP or CyI (CyI is 5.0 脳 10-5mol/L to label BMSCs;-2 safely and effectively. The optical imaging of BMSCs labeled by RFP or CyI is feasible in F344 rats. 3. In vitro imaging can be used to trace the local injection of BMSCs;-4 into the heart. The BMSCs. labeled by USPIO can still be traced in vivo 2 weeks after labeling.
【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】：2012
【分类号】：R329.2
本文编号：2461808
[Abstract]:Objective: to study the safety and efficacy of F344 rat bone marrow mesenchymal stem cells (BMSCs) labeled with red fluorescent protein (RFP) and fluorescent dye CyI (derivatives of Cy5). The feasibility of imaging the labeled stem cells on the surface of the rat was studied, and the possibility of bimodal labeled stem cell tracer imaging in vivo was explored by combining ultra-small superparamagnetic iron oxide particles with (USPIO) and labeling BMSCs, simultaneously. Methods: F344 rat BMSCs (BMSCs/RFP) expressing RFP stably was successfully transfected and co-cultured with BMSCs/RFP with different concentration of CyI for 24 h. In vitro, the fluorescence intensity was measured by fluorescence detector, and the imaging effect of F344 rats injected with optically labeled cells on the body surface was detected by optical imaging system. The medium containing USPIO (40 渭 g / ml) and polylysine (PLL, 1.5 渭 g / ml) was incubated with BMSCs/RFP for 24 h. The distribution of iron particles in the cells was observed by Prussian blue staining. Optical imaging of rat body surface was performed by subcutaneous or intramuscular injection of RFP or CyI labeled BMSCs,. In vivo, a F344 rat model of acute myocardial infarction was established by ligating the left anterior descending branch of the coronary artery (LAD) by thoracotomy. Three kinds of labeled cells (BMSCs/RFP,CyI or USPIO labeled BMSCs/RFP, in which USPIO/RFP was double labeled) were injected into the myocardium of F344 rats. Optical imaging and MRI were used to label the cells in vivo. Prussian blue staining and fluorescence imaging were used to observe the distribution of USPIO particles and RFP in heart. Results: in vitro fluorescence intensity analysis showed that BMSCs labeled with RFP or CyI (5.0 脳 10-5mol/L in CyI) had a strong signal intensity when the cell concentration was higher than 5.0 脳 105/ml. When the concentration of CyI and BMSCs/RFP were 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L respectively, the cell morphology and the number of dead cells did not change significantly at the concentration of 5.0 脳 10 ~ (- 6), 1.0 脳 10 ~ (- 5) and 5.0 脳 104mol/L. When the number of injected cells was 1.0 脳 10 6, the fluorescence imaging effect of CyI was better than that of RFP. in rat body surface imaging. The acute myocardial infarction model of F344 rats was successfully established by ligating the left anterior descending coronary artery (LAD) under ventilator-assisted ventilation. Labeled cells injected locally into the myocardium did not detect fluorescence signals in vivo imaging under optical conditions. In vitro imaging was able to detect weak fluorescence on the heart surface: the signal intensity of the injection region was significantly decreased by MRI within 2 weeks after labeling. Pathological Prussian blue staining and fluorescence imaging suggested that the distribution of iron particles and RFP in myocardium was consistent with that of stem cells transplantation. Conclusion: 1. The concentration of RFP or CyI (CyI is 5.0 脳 10-5mol/L to label BMSCs;-2 safely and effectively. The optical imaging of BMSCs labeled by RFP or CyI is feasible in F344 rats. 3. In vitro imaging can be used to trace the local injection of BMSCs;-4 into the heart. The BMSCs. labeled by USPIO can still be traced in vivo 2 weeks after labeling.
【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】：2012
【分类号】：R329.2
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