超顺磁性氧化铁微粒磁标记骨髓间充质干细胞效率实验研究

目的探讨不同浓度超微超顺磁性氧化铁微粒（USPIO）与多聚左旋赖氨酸（PLL）标记大鼠骨髓问充质十细胞（BMSCs）的磁标记效率及对细胞生长活力的影响，寻找最佳配比浓度。方法实验采用6周龄Wister近交系大鼠，150g芹右．雄性。用贴壁法分离培养BMSCs。使用6nmUSPIO—PLL复合物标记BMSCs。实验分6组，对照组为未标记的BMSCs（A组）。按照PLL的有无及PLL质量浓度梯度（0、0．25、0．50、0．75、1．00μg／mL）分为5个实验组（B～F组）；其中每个实验组再根据不同浓度铁离子与PLL结合（USPIO的终质量浓度分别为25、50、100、150μg/mL）。使用电子显微镜及光学显微镜观察标记后的BMSCs及BMSCs内的USPIO微粒。BMSCs活力测定采用台盼蓝排除实验。BMSCs生长曲线绘制采用MTT法。MRI观察体外标记后BMSCs的显影。火焰法测量BMSCs内铁含量，验证铁含量与MRI信号的关系。统计学分析采用方差分析。，结果台盼蓝染色证实90％以上标记后BMSCs均拒染台盼蓝。根据BMSCs生长曲线判断单纯使用铁离子质量浓度达到200μg／mL时或PLL用量达1．00μg／mL会对BMSCs的生长产生一定的抑制作用。火焰法测得单独USPIO标记BMSCs与USPIO—PLL标记BMSCs铁含量差异有统计学意义（P〈0．05）。T2WI及SWI序列图像从B组至F组信号强度逐级下降，反映出铁离子的变化情况。结论使用USPIO（100μg／mL）结合PLL（0．75μg／mL）标记BMSCs即对细胞活性和生长无不利影响．且标记BMSCs的信号强度较强。

Experimental study on magnetic labeling of bone marrow mesenchymal stem cells with super paramagnetic iron oxideparticles

Objective To explore the efficiency of magnetic labeling of bone marrow mesenchymal stem cells（BMSCs） with super paramagnetic iron oxide particles（USPIO） and its impact on cell growth. Methods Six-week-old male Wister inbred rat with body weight of 150 g was sacrificed. BMSCs were isolated and cultured by adherent method, and labeled with 6 nm USPIO-PLL complexes. Six groups of cells were set up： the unlabeled BMSCs were used as control group （group A）, and the other cells were divided into 5 experimental groups（group B to F） accorded to the presence or absence of PLL and PLL concentration gradient（0, 0.25, 0.50, 0.75, 1.00 μg/mL）. In each experimental group, Fe ions of different concentrations were bond to PLL（final concentrations of USPIO were 25, 50, 100, and 150 μg/mL, respectively）. The labeled BMSCs and intracellular USPIO particles were observed with electron microscope and light microscope, the cell viability was determined by Trypan blue exclusion test, and the cell growth curve was observed by methyl thiazoly tetrazolium salt（MTT） assay. Magnetic resonance imaging （MR1） was employed to image the labeled cells. Flame method was used to measure the iron content within BMSCs and verify the relationship between the iron content amt MRI signal, and variance analysis was made for statistical analysis. Results Over 90 % of the labeled cells were resistant to Trypan blue staining. Accorded to the cell growth curve, the ions at concentration of 200 μg/mL or PLL at 1.00 μg/mL inhibited the cell growth to certain extent. As measured by flame method, significant difference in iron contents was observed between the ceils labeled with USPIO and cells labeled with USPIO plus PLL（P 〈 0.05）. Signal intensities of the images of sequences T2WI and susceptibility weighted images（SW1） decreased progressively from group B to group F, which reflected the changes in Fe ions. Conclusion It is demonstrated that labeling BMSCs with USPIO（100 μg/mL） combined with PLL（0.75 μg/mL） will not adversely affect cell viability and growth, and the signal intensities of the labeled cells are strong.