摘要
目的观察超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)标记的自体BMSCs在兔椎间盘内的存活时间,为后期采用转染成功的BMSCs预防椎间盘退变提供依据,并观察其迁移规律。方法8～10周龄日本大耳白兔12只,雌雄不限,体重1.5～2.0kg,取4mL骨髓密度梯度离心法行BMSCs分离、培养。取第3代BMSCs(1×106个)用SPIO(25μg/mL)标记,普鲁士蓝铁染色检测标记效率,透射电镜观察标记细胞,锥虫蓝染色鉴定标记细胞活力,MTT法测定标记细胞及未标记细胞增殖,并绘制生长曲线。将12只兔随机分为实验组(n=8)和对照组(n=4),分别于L1、2、L2、3、L3、4、L4、5注入标记和未标记自体BMSCs悬液20μL(细胞浓度均为5×105个/mL),术后2、4、6、8周实验组和对照组分别取2只和1只动物处死,取相应椎间盘行普鲁士蓝铁染色观察细胞是否存活以及分布。结果第3代BMSCs经SPIO标记后,普鲁士蓝铁染色标记效率为95.65%±1.06%;透射电镜观察示细胞质内有电子高密度铁颗粒;锥虫蓝染色示细胞活力为98.28%±0.85%;MTT检测显示7d内未标记细胞组和标记细胞组间细胞增殖比较差异无统计学意义(P>0.05)。实验组细胞移植术后8周,仍可观测到位于细胞质内的蓝染颗粒。对照组未见蓝染颗粒。结论SPIO可以安全、有效地标记BMSCs,并能在体内持续存活8周。
Objective To explorer the survival time of autogeneic BMSCs labeled by superparamagnetic iron oxide （SPIO） in rabbit intervertebral discs and the rule of migration so as to prove bases of gene therapy preventing intervertebral disc degeneration. Methods Twelve rabbits were used in this experiment, aged 8-10 weeks, weighing 1.5-2.0 kg and neglecting their gender. BMSCs were separated from rabbits bone marrow by density gradient centrifugation and cultivated, and the 3rd generation of BMSCs were harvested and labeled with SPIO, which was mixed with poly-l-lysine. The labeling efficiency was evaluated by Prussian blue staining and transmission electron microscope. Trypanblan stain and MTT were performed to calculate the cell＇s activity. Rabbits were randomly divided into experimental group （n=8） and control group （n=4）, the labeled BMSCs and non-labeled BMSCs （5×10^5/mL） were injected into their own intervertebral discs （L1,2, L2.3, L3.4 and L4.5）, respectively. At 2, 4, 6 and 8 weeks, the discs were treated with Perl＇s fluid to observe cell survival and distribution. Results The labeling efficiency of BMSCs with SPIO was 95.65% ± 1.06% , the cell activity was 98.28% ± 0.85%. There was no statistically significant difference in cell proliferation within 7 days between non-labeled and labeled cells （P 〉 0.05）. After 8 weeks of operation, the injected cells was alive. Conclusion Labeled BMSCs with SPIO is feasible in vitro and in vivo, and the cells can survive more than 8 weeks in rabbit discs.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2009年第11期1355-1359,共5页
Chinese Journal of Reparative and Reconstructive Surgery
关键词
BMSCS
超顺磁性氧化铁标记
细胞移植
细胞迁移
椎间盘退变
兔
BMSCs Superparamagnetic iron oxide label Cell transplantation Cell migration Intervertebral disc degeneration Rat
