超顺磁性氧化铁和绿色荧光蛋白双标骨髓间充质干细胞体外诱导类神经元样分化的生物学活性及MRI研究  

Research on biological activities and MRI of differentiations of neural-like cells induced by superparamagnetic iron oxide and green fluorescent protein double-labeled bone marrow-derivedmesenchymal stem cells in vitro

作　　者：吴莉[1] 赵卫[1] 陈泽谷[3] 何波[1] 陆琳[1] 赵娴[2] 刘流[2] 

机构地区：[1]昆明医科大学第一附属医院医学影像科,650032 [2]昆明医科大学第一附属医院整形外科 [3]中南大学湘雅医学院附属海口医院放射科

出　　处：《中华放射学杂志》2016年第3期217-222,共6页Chinese Journal of Radiology

基　　金：国家自然科学基金（81460298）;云南省科技厅-昆明医科大学应用基础研究联合专项（2015FB038）

摘　　要：目的探讨超顺磁性氧化铁（SPIO）及绿色荧光蛋白（GFP）双标的骨髓间充质干细胞（BMSCs）向类神经元样分化的生物学活性及MRI信号强度fsI）变化特点。方法将GFP-BMSCs体外标记不同浓度SPIO（A～D组铁浓度分别为25、50、75、100μg／ml，E组未标SPIO为对照组），普鲁士蓝染色检测各组SPIO标记率，原子分光光度计测量铁含量，CCK8实验检测细胞增殖率，PCR检测细胞周期；A～E组均将1×10^8个细胞置试管内行MRT2＊WI、磁敏感加权成像（SWI），测量2个序列图像sI变化并进行比较，找出最佳成像浓度组与E组共同诱导类神经元样分化，72h行茜素红、骨钙素及尼氏、神经元特异性核蛋白（NeuN）染色、神经丝蛋白200染色，实时定量PCR检测β-Ⅲ微管蛋白ftub3）、巢蛋白（Nestin）、神经元特异性烯醇化酶（NSE）、微管相关蛋白2（MAP．2）、突触结合蛋白（Syt1）RNA表达量，比较两组细胞染色阳性率及RNA表达量，最终采用SWl分析SI差异。多组间比较用单因素方差分析，多组间两两比较用LSD-t检验；两组均数比较采用独立样本t检验。结果A～D组SPIO标记率为100％，A～E组细胞内铁含量分别为（14．36±7．61）、（21．73±3．42）、（30．54±8．73）、（33．65±9．62）、（2．31±0．32）pg／cell，A～D组均较E组高（F=3．852，P=0．003），C组与D组间差异无统计学意义fP=0．267）；CCK8实验4值D组最小（3．18±O．46）；A～E组sI变化SWI序列均较T2＊WI序列低，差异均有统计学意义。SWI序列SI变化C组（145．89±14．31）与D组（127．37±12．21）差异无统计学意义fP=0．064），其余各组两两比较差异均有统计学意义（P值均〈0．01）。C组SWI和T2＊WISI衰减百分率分别为48．15％、69．34％。C、E2组诱导向类神经元样分化，非神经元细胞比例、尼氏染色细胞阳性率比较差异均无统计学意义俨值均〉0．05）。tuObjective To explore the biological activities and the MR imaging signal intensities （Sis） characteristics of differentiations of neural-like cells induced by superparamagnetic iron oxide （SPIO） and green fluorescent protein （GFP） double-labeled bone marrow-derived mesenchymal stem cells （BMSCs） in vitro. Methods GFP-BMSCs were labeled with different concentrations of SPIO in vitro （the concentration of the A, B, C and D group was 25, 50, 75 and 100 ug/ml, respectively; the E group without labels of SPIO served as the control group）. The Prussian blue stainings were used to detect the labeling rates of SPIO. The iron contents of cells were measured by atomic absorption spectrometer. The CCK8 experiments were used to detect the cell proliferation rates. The cell cycles were detect by PCR. Each of the A-E groups had a test tube with 1× 108 cells. All test tubes underwent T2＊ weighted imaging （T2＊WI） and susceptibility weighted imaging （SWI） in a MR imaging scanner. The optimal group was defined by comparing the measurements of Sis between T2＊WI and SWI. The optimal group and the E group together induced the differentiations of osteogenesis and neural-like ceils. The stainings of alizarin red, osteocalein and Nissl, NeuN, and NF-200 were performed at 72 hours. Real-time quantitative PCR was used to detect the expression levels of RNA in tub3, nestin, NSE, MAP-2 and Sytl. The positive staining rates and the expression levels of RNA were compared between the two groups. Finally, SWI was used to analyse the changes of Sis. One-way analysis of variance （ANOVA） was used to the multi-group comparison. Using least significant difference （LSD） test to analyse the comparisons between the multi-groups. Results The labeling rates of the A-D groups were 100%. The iron contents of ceils in the A-E groups were （14.36± 7.61）, （21.73±3.42）, （30.54±8.73）, （33.65±9.62）, and （2.31±0.32） pg/cell, respectively. The iron contents of cells in the A-D groups were significa

关 键 词：充质干细胞 磁共振成像 分子探针 

分 类 号：R445.2[医药卫生—影像医学与核医学]