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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)标记大鼠骨髓间充质干细胞(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,150 g左右,雄性。用贴壁法分离培养BMSCs。使用6nm USPIO-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。
Objective To investigate the magnetic labeling efficiency and cell viability of rat bone marrow mesenchymal stem cells (BMSCs) labeled with different concentrations of ultramicro-paramagnetic iron oxide particles (USPIO) and poly-L-lysine (PLL) Good ratio of concentration. Methods Six weeks old Wister inbred rats, about 150 g, were males. BMSCs were isolated and cultured by adherent method. BMSCs were labeled with a 6 nm USPIO-PLL complex. The experiment was divided into 6 groups, the control group was unlabeled BMSCs (group A). According to the presence or absence of PLL and PLL gradient (0,0.25,0.50,0.75,1.00μg / mL) divided into five experimental groups (B ~ F group); each experimental group according to different concentrations of iron ions and PLL (USPIO final concentration of 25,50,100,150μg / mL). The labeled BMSCs and USPIO particles in BMSCs were observed under an electron microscope and light microscope. BMSCs viability assay using trypan blue exclusion experiment. BMSCs growth curve drawn using MTT method. MRI observation of the development of BMSCs after in vitro labeling. Flame method to measure the content of iron in BMSCs to verify the relationship between iron content and MRI signal. Statistical analysis using analysis of variance. Results Trypan blue staining confirmed that more than 90% of labeled BMSCs were labeled with trypan blue. According to the growth curve of BMSCs, the growth of BMSCs was inhibited when pure iron concentration was 200μg / mL or PLL was 1.00μg / mL. Flame method measured USPIO-labeled BMSCs and USPIO-PLL labeled BMSCs iron content was statistically significant (P <0.05). The signal intensity of T2WI and SWI sequence from B to F decreased step by step, reflecting the change of iron ion. Conclusion BMSCs labeled with USPIO (100μg / mL) combined with PLL (0.75μg / mL) had no adverse effect on cell viability and growth, and the signal intensity of labeled BMSCs was stronger.