Fe3O4纳米化颗粒标记人羊膜间充质干细胞的合适条件

Optimal condition of Fe_3O_4 nanoparticles labeling to human amniotic-derived mesenchymal stem cells

目的:Fe3O4纳米化颗粒是近来发现的活体示踪剂,由于其毒性可导致标记细胞死亡,寻找合适的浓度进行细胞标记已成为活体示踪的关键。实验以人羊膜间充质干细胞作为靶点,探讨Fe3O4纳米化颗粒标记的合适条件。方法:实验于2007-08在郑州大学完成。①细胞来源及颗粒:人羊膜间充质干细胞由郑州大学第一临床医学院神经外科杨波教授自健康产妇胎盘羊膜中提取,产妇对实验知情同意,实验经医院医学伦理委员会批准。Fe3O4纳米颗粒由美国Sigma公司生产,商品名:菲立磁,批号094k0788。转染剂多聚左旋赖氨酸购自大连宝生生物公司,批号033K4351。②实验方法:向人羊膜间充质干细胞加入含10%胎牛血清、20μg/L碱性成纤维生长因子的DMEM/F12培养基,置于37℃、体积分数为0.05的CO2饱和湿度培养箱中培养,待细胞达80%～90%融合时胰酶消化传代。取传至第3代的细胞,放入含DMEM/F12培养基的10mL培养瓶中,密度调整为1×109L-1。设立3组:单纯Fe3O4组分为4个亚组,即向培养基中分别加入终浓度为20,30,40,80mg/L的Fe3O4纳米颗粒;Fe3O4+多聚左旋赖氨酸组分为4个亚组,即向培养基中分别加入上述终浓度Fe3O4纳米颗粒后,再加入1.5mg/L多聚左旋赖氨酸;培养基对照组,仅加入DMEM/F12培养基。各组细胞标记12h后,均更换为DMEM/F12培养基培养3周。③实验评估:分别于标记后12h、36h、1周和3周,普鲁士蓝染色检测Fe3O4纳米颗粒进入细胞情况,锥虫蓝染色检测细胞活性。结果:①Fe3O4纳米颗粒标记人羊膜间充质干细胞的效果:终浓度为20mg/L的Fe3O4纳米颗粒细胞标记率为60%,终浓度为30,40,80mg/L的Fe3O4纳米颗粒细胞标记率均为100%。单纯Fe3O4组可见少量蓝染的Fe3O4纳米化颗粒分散于细胞浆内,Fe3O4+多聚左旋赖氨酸组蓝染颗粒明显增多,部分聚集成团。②标记后细胞活性测定:与培养基对照组比较,当Fe3O4终浓度为20,30mg/L时,单纯Fe3O4组、Fe3O4+多聚左旋赖氨酸组细胞活性均无明显变化(P>0.05);当Fe3O4终浓度升高至40,80mg/L时,上述2组细胞活性均显著降低(P<0.05)。结论:①与多聚左旋赖氨酸混合,能够使进入细胞内的Fe3O4纳米颗粒增多,从而加强细胞标记效果。②30mg/LFe3O4纳米颗粒细胞标记率可达100%,且该浓度不影响人羊膜间充质干细胞的生物活性。③30mg/LFe3O4纳米颗粒联合多聚左旋赖氨酸是标记人羊膜间充质干细胞的合适条件。

AIM： Fe3O4 nanoparticle is one kind of labeled agents in vivo, which is discovered recently. Owing to its fatal toxicity to labeled cells, it is vital to find the appropriate concentration when labeling cells in vivo. Therefore, this study chose human amniotic-derived mesenchymal stem cells （AD-MSCs） as target in order to explore the best condition for labeling human AD-MSCs by Fe3O4 nanoparticle. METHODS： The experiment was completed in Zhengzhou University in August 2007.1.The source of cells and particles： Human AD-MSCs were extracted from the placenta amniosis of healthy parturient by professor Yang Bo working in the First Affiliated Hospital of Zhengzhou University. All the parturients signed informed consents. Moreover, the experiment was approved by the Medical Ethical Committee of the hospital. Fe3O4 nanoparticle was produced by Sigma corporation in America, titled Ferric Oxide Particles, with 094k0788 as its Batch No. Poly L lysine （PLL）, the transfection agent, was produced by TAKARA Biotechnology （Dalian） CO.,Ltd, with 033K4351 as its Batch No.2.Empirical methods： AD-MSCs were added to nutritive medium DMEM/F12 which contained 10% calf serum and 20 μg/L basic fibroblast growth factor. Cells were then cultivated in an incubator of saturated humidity at 37℃, with 0.05 volume fraction of CO2 nutritive medium. When 80%-90% cells were mixed, trypsinization and was underwent and cells were subcultured. The cells in the third generation were put into 10 mL culture flask containing nutritive medium DMEM/F12. The density was adjusted to 1 × 10^9 L^-1. Those cells were divided into 3 groups： The group of pure Fe3O4 was further divided to 4 sub-groups, adding with Fe3O4 nanoparticles at different final concentrations of 20, 30, 40, 80 mg/L respectively to nutrient medium. The group of the mixture of Fe3O4 nanoparticle and PLL was also further divided into 4 sub-groups, adding 1.5 mg/L PLL to nutrient medium besides adding the same stuff as the first group. Control group of nutrient medium that was only added with DMEM/F12. After labeling cells in those groups for 12 hours, they were cultivated in nutritive medium DMEM/F12 for 3 weeks.3.Experiment evaluation： Prussian blue staining method was adopted to observe the condition of Fe3O4 nanoparticle＇s entry to cells 12 hours, 36 hours, 1 week and 3 weeks later. Trypan blue dying method was adopted to detect cytoactivity. RESULTS： 1.The effect of Fe3O4 nanoparticle labeling to AD-MSCs： The labeling rate of Fe3O4 nanoparticle at the final concentration of 20 mg/L was 60%, and the labeling rates at the final concentration of 30, 40, 80 mg/L were all 100%. Few blue-stained Fe3O4 nanoparticles were scattered among cytolymphs in the group of pure Fe3O4 nanoparticles. The blue-stained Fe3O4 nanoparticles were obviously increased in the group of the mixture of Fe3O4 nanoparticles and PLL, with some of them gathering into conglobation.2.The detection of cytoactivity after labeling： Compared with the control group, the cytoactivity of the first two groups had no significant difference when Fe3O4 nanoparticles were at the final concentrations of 20 and 30 mg/L （P 〉 0.05）; Yet when the final concentration increased to 40 and 80 mg/L, the cytoactivity of the first two groups decreased significantly （P 〈 0.05）. CONCLUSION： 1.Mixing PLL with Fe3O4 nanoparticles can make more Fe3O4 nanoparticles enter into cells so as to strengthen the labeling effect of cells.2.The labeling effect of Fe3O4 nanoparticles at the concentration of 30 mg/L can reach 100%, and this concentration does not affect the bioactivity of AD-MSCs.3.The mixture of Fe3O4 nanoparticles and PLL at the concentration of 30 mg/L is the best condition for labeling AD-MSCs.