兔脂肪间充质干细胞的培养鉴定及体外磁标记MR成像  

Culture and identification of rabbit adipose-derived stem cells and its magnetic resonance imaging in vitro

作　　者：谢光友[1] 杨海涛[1] 吕富荣[1] 鲁文力 盛波[1] 肖智博[1] 吕发金[1] 欧阳羽[1] 

机构地区：[1]重庆医科大学附属第一医院放射科,重庆400016

出　　处：《第三军医大学学报》2014年第15期1567-1571,共5页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2011jjA10082)~~

摘　　要：目的研究兔脂肪间充质干细胞(adipose derived stem cells,ADSCs)的培养鉴定方法,联合应用超顺磁性氧化铁(superparamagnetic iron oxide particles,SPIO)和多聚赖氨酸(poly-l-lysine,PLL)行体外磁标记细胞,探讨磁标记示踪的可行性。方法分离、纯化并培养兔ADSCs,流式细胞术鉴定细胞表面抗原CD90、CD44和CD34。应用SPIO-PLL标记细胞,普鲁士蓝染色和透射电镜检测胞内铁粒子。体外3.0T MR分别对1×107个未标记ADSCs(空白对照),经25、50、75μg/mL SPIO-PLL分别标记的1×107个细胞,经25μg/mL SPIO-PLL标记的1×107个(标记1 d)、1×107个(标记3 d)和5×106个(标记1 d)ADSCs进行成像,包括T1WI、T2WI及T*2WI序列,测量各组的信号强度和弛豫时间。结果第3代ADSCs纯度高、排列规则,细胞呈漩涡状,流式细胞术检测结果示CD44和CD90阳性表达率分别为99.2%、98.7%,CD34表达阴性。普鲁士蓝染色和透射电镜示胞浆内蓝色颗粒,磁标记率近100%。MR示随着SPIO-PLL浓度增高,T*2WI和T2WI序列的信号强度变化较T1WI明显。1×107个ADSCs(标记1 d)的信号强度变化率较1×107个(标记3 d)和5×106个(标记1 d)大,T*2和T2弛豫时间与T1弛豫时间相比差异均有统计学意义(F=161.47,P<0.05),但T*2和T2弛豫时间相比差异无统计学意义(F=5.88,P>0.05)。结论通过分离纯化培养可获得足量的ADSCs,SPIO-PLL能够有效标记ADSCs,体外可行MR细胞成像,以T*2WI和T2WI序列敏感。Objective To investigate how to culture and identify rabbit adipose-derived stem cells （ADSCs）, and to explore the feasibility of tracing ADSCs after being labeled by superparamagnetic iron oxide particles （SPIO） combined with poly-l-lysine （PLL） in vitro. Methods ADSCs were isolated from New Zealand rabbit, and then purified and primarily cultured. Flow cytometry （FCM） was used to identify cell surface antigens CIM4, CD90 and CD34. Transmission electron microscopy （TEM） was used to observe the intracellular iron particles in SPIO-PLL labeled ADSCs after Prussian blue staining. 3.0-T MR scanning in vitro system was used to scan 1 × 107 ADSCs unlabeled and labeled with 25, 50 and 75 μg/mL SPIO-PLL respectively, or labeled with 25 μg/mL for 1 or 3 d, or to scan 5 × 106 cells labeled with 25 μg/mL for 1 d. T1WI, T2WI and T2＊WI arrays were performed for the signal intensity （SI） and relaxation time of each tube. Results The ADSCs of third generation were of high purity, in regular arrangement presenting whirlpool-like. The cells had an expression rate of 98.7% for CD44 and 99.2% for CD90, but negative expression for CD34. TEM showed that iron particles existed in the cytoplasm of ADSCs after Prussian blue staining, and the rate of labeling was nearly 100%. T2＊ WI and T2WI demonstrated a more obvious decrease in SI than that of T1WI with the increasing concentration of SPIO-PLL. The percentage of SI in 1 ×107 ADSCs （labeled for 1 d） was significantly higher than that of 1 × 107 （labeled for 3 d） and 5 × 106（labeled for 1 d）. The relaxation time of T2＊ and T22 compared with T1 was significant different （F = 161.47, P 〈 0.05 ）, but the relaxation time between T2＊ and T2 was not statistically significant （ F = 5.88, P 〉 0.05 ）. Conclusion Sufficient ADSCs are obtained through isolation, purification and culture. ADSCs are labeled effectively by SPIO-PLL and detected through MR scanning in vitro, with T2＊WI and T2WI arrays more sensitive.

关 键 词：脂肪间充质干细胞 超顺磁性氧化铁 磁标记 磁共振成像 体外 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R392.33[医药卫生—基础医学]