超小超顺磁性氧化铁（USPIO）标记SD大鼠脂肪来源干细胞（ADSCs）体外和体内实验研究
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摘要
目的：利用超小超顺磁性氧化铁颗粒(USPIO)标记SD大鼠脂肪来源干细胞(ADSCs),探讨标记的有效性及安全性,并从体外和体内水平探究MR对标记细胞进行成像示踪的可行性。
方法：使用含USPIO (40μg/ml)和多聚赖氨酸(PLL,1.5μg/ml)的培养基与ADSCs共孵育培养24h,标记后行普鲁士蓝染色和透射电镜观察铁颗粒在细胞内的分布,行台盼蓝染色和MTS实验测定标记对细胞活力和增殖能力的影响,应用ELISA法比较标记细胞和对照组培养液中血管内皮生长因子(VEGF)含量。体外条件下,通过T2map和T2*map序列研究不同数量标记细胞与MR信号参数(T2、T2*、R2和R2*)之间相关性。活体条件下,开胸结扎冠脉前降支(LAD)建立SD大鼠急性心梗模型,通过尾静脉和心肌局部注射分别移植标记细胞,利用MR进行标记细胞的活体示踪成像。取病理行普鲁士蓝染色观察USPIO颗粒在各脏器分布情况。
结果：体外普鲁士蓝染色显示USPIO标记ADSCs的阳性率为99%,透射电镜提示USPIO颗粒主要位于胞质内溶酶体中。台盼蓝染色实验显示活细胞数大于95%,MTS实验提示USPIO浓度为10、20、40、80和160μg/ml时不影响ADSCs增殖。ELISA法显示标记细胞和对照组培养液中VEGF含量与细胞数量呈正相关,组间无显著差异。体外MR成像可敏感显示USPIO标记细胞,通T2map序列及T2*map序列可以进行T2和T2*定量测量,R2和R2*值与标记细胞数量之间呈线性相关。呼吸机辅助通气条件下通过开胸结扎冠脉前降支(LAD)可成功建立SD大鼠急性心梗模型。尾静脉途径移植标记细胞可在肝脏首先观察到信号强度减低,心肌信号强度无显著改变；通过心肌直接注射标记细胞可在MR观察到注射区域信号强度明显降低。普鲁士蓝染色提示两种标记途径可在梗死心肌附近发现局灶性分布的USPIO颗粒,但大部分颗粒分布于脾脏中。
结论：
1、使用USPIO (40μg/ml)和PLL(1.5μg/ml)可以安全、有效地标记ADSCs;
2、USPIO标记对ADSCs分泌VEGF没有影响；
3、体外条件下MR可敏感显示USPIO标记细胞,R2及R2*与细胞数量呈线性相关。
4、MR活体示踪USPIO标记ADSCs的敏感性与移植途径相关。
Objective:To investigate the efficacy and safety of ultrasmall superparamagnetic iron oxide (USPIO) labeling adipose derived stem cells (ADSCs) of SD rats, and the feasibility of tracing labeled cells with MR imaging was studied both in vitro and in vivo.
Methods and Materials:ADSCs were incubated with culture medium containing40μg/ml USPIO and1.5ug/ml poly-1-lysine (PLL) for24h. The distribution of iron particles in cells was studied by Prussian blue staining and transmission electron microscopy(TEM). Besides, trypan-Blue stain and MTS were used to determine the cell viability and proliferation. ELISA assay was used to compare the vascular endothelial growth factor (VEGF) levels in culture medium between labeled cells and control group. In vitro, T2map and the T2*map sequence were selected to study the correlation between the number of labeled cells and MR signals (T2, T2*, R2and R2*). In vivo, SD rat model of acute myocardial infarction was established by ligating the anterior descending coronary artery(LAD). The labeled ADSCs were transplanted through both the tail vein and myocardial direct injection, then MR was performed to trace the cells. Postmortal study was carried out to observe the distribution of USPIO particles in the various organs with Prussian blue stain.
Results:After incubating the cells with USPIO and PLL for24h, the percentage of labeled ADSCs reached up to99%. Iron particles inside the cells were confirmed by TEM, which mainly in lysosomes. Trypan-Blue stain showed the number of living cells was greater than95%. MTS experiments suggest that USPIO (10,20,40,80,160μg/ml) exert no significant influence on the proliferation of ADSCs. ELISA assay revealed VEGF levels in culture medium were positively correlated with the number of cells. No significant difference was found between labeled cells and the control group. MR imaging was sensitive detecting USPIO labeled cells in vitro, T2and T2*value could be quantitatively measured by T2map and T2*map sequences. In addition, linear correlations between cell numbers and R2or R2*values were discovered. Under mechanical ventilation, the SD rats model of acute myocardial infarction was successfully established by ligating anterior descending coronary artery (LAD). The signal of liver decreased after intravenous injection of labeled cells. The signal intensity in myocardium had no significant change through intravenous injection; while it decreased significantly when labeled cells were injected directly into myocardium. Prussian blue staining diplayed only a few USPIO particles in the infracted myocardium, the major part however, distributed in spleen.
Conclusion:
1. Using USPIO(40μg/ml) and PLL(1.5μg/ml) could safely and effectively label ADSCs
2. USPIO labeling had no significant effect on VEGF secreted by ADSCs
3. MRI could sensitively detect USPIO labeled cells in vitro, with a positive linear correlation between R2and R2*values and cell numbers.
4. The sensitivity of detection of USPIO-labeld ADSCs in vivo by MRI was influenced by transplanted pathway.
方法：使用含USPIO (40μg/ml)和多聚赖氨酸(PLL,1.5μg/ml)的培养基与ADSCs共孵育培养24h,标记后行普鲁士蓝染色和透射电镜观察铁颗粒在细胞内的分布,行台盼蓝染色和MTS实验测定标记对细胞活力和增殖能力的影响,应用ELISA法比较标记细胞和对照组培养液中血管内皮生长因子(VEGF)含量。体外条件下,通过T2map和T2*map序列研究不同数量标记细胞与MR信号参数(T2、T2*、R2和R2*)之间相关性。活体条件下,开胸结扎冠脉前降支(LAD)建立SD大鼠急性心梗模型,通过尾静脉和心肌局部注射分别移植标记细胞,利用MR进行标记细胞的活体示踪成像。取病理行普鲁士蓝染色观察USPIO颗粒在各脏器分布情况。
结果：体外普鲁士蓝染色显示USPIO标记ADSCs的阳性率为99%,透射电镜提示USPIO颗粒主要位于胞质内溶酶体中。台盼蓝染色实验显示活细胞数大于95%,MTS实验提示USPIO浓度为10、20、40、80和160μg/ml时不影响ADSCs增殖。ELISA法显示标记细胞和对照组培养液中VEGF含量与细胞数量呈正相关,组间无显著差异。体外MR成像可敏感显示USPIO标记细胞,通T2map序列及T2*map序列可以进行T2和T2*定量测量,R2和R2*值与标记细胞数量之间呈线性相关。呼吸机辅助通气条件下通过开胸结扎冠脉前降支(LAD)可成功建立SD大鼠急性心梗模型。尾静脉途径移植标记细胞可在肝脏首先观察到信号强度减低,心肌信号强度无显著改变；通过心肌直接注射标记细胞可在MR观察到注射区域信号强度明显降低。普鲁士蓝染色提示两种标记途径可在梗死心肌附近发现局灶性分布的USPIO颗粒,但大部分颗粒分布于脾脏中。
结论：
1、使用USPIO (40μg/ml)和PLL(1.5μg/ml)可以安全、有效地标记ADSCs;
2、USPIO标记对ADSCs分泌VEGF没有影响；
3、体外条件下MR可敏感显示USPIO标记细胞,R2及R2*与细胞数量呈线性相关。
4、MR活体示踪USPIO标记ADSCs的敏感性与移植途径相关。
Objective:To investigate the efficacy and safety of ultrasmall superparamagnetic iron oxide (USPIO) labeling adipose derived stem cells (ADSCs) of SD rats, and the feasibility of tracing labeled cells with MR imaging was studied both in vitro and in vivo.
Methods and Materials:ADSCs were incubated with culture medium containing40μg/ml USPIO and1.5ug/ml poly-1-lysine (PLL) for24h. The distribution of iron particles in cells was studied by Prussian blue staining and transmission electron microscopy(TEM). Besides, trypan-Blue stain and MTS were used to determine the cell viability and proliferation. ELISA assay was used to compare the vascular endothelial growth factor (VEGF) levels in culture medium between labeled cells and control group. In vitro, T2map and the T2*map sequence were selected to study the correlation between the number of labeled cells and MR signals (T2, T2*, R2and R2*). In vivo, SD rat model of acute myocardial infarction was established by ligating the anterior descending coronary artery(LAD). The labeled ADSCs were transplanted through both the tail vein and myocardial direct injection, then MR was performed to trace the cells. Postmortal study was carried out to observe the distribution of USPIO particles in the various organs with Prussian blue stain.
Results:After incubating the cells with USPIO and PLL for24h, the percentage of labeled ADSCs reached up to99%. Iron particles inside the cells were confirmed by TEM, which mainly in lysosomes. Trypan-Blue stain showed the number of living cells was greater than95%. MTS experiments suggest that USPIO (10,20,40,80,160μg/ml) exert no significant influence on the proliferation of ADSCs. ELISA assay revealed VEGF levels in culture medium were positively correlated with the number of cells. No significant difference was found between labeled cells and the control group. MR imaging was sensitive detecting USPIO labeled cells in vitro, T2and T2*value could be quantitatively measured by T2map and T2*map sequences. In addition, linear correlations between cell numbers and R2or R2*values were discovered. Under mechanical ventilation, the SD rats model of acute myocardial infarction was successfully established by ligating anterior descending coronary artery (LAD). The signal of liver decreased after intravenous injection of labeled cells. The signal intensity in myocardium had no significant change through intravenous injection; while it decreased significantly when labeled cells were injected directly into myocardium. Prussian blue staining diplayed only a few USPIO particles in the infracted myocardium, the major part however, distributed in spleen.
Conclusion:
1. Using USPIO(40μg/ml) and PLL(1.5μg/ml) could safely and effectively label ADSCs
2. USPIO labeling had no significant effect on VEGF secreted by ADSCs
3. MRI could sensitively detect USPIO labeled cells in vitro, with a positive linear correlation between R2and R2*values and cell numbers.
4. The sensitivity of detection of USPIO-labeld ADSCs in vivo by MRI was influenced by transplanted pathway.
引文
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