Fe3O4 纳米化颗粒标记人羊膜间充质干细胞的合适条件**

王公平1-3，杨波1，关方霞4，杜英5，常克亮1，宋来君1，胡祥6，曾光伟2

课题背景：课题获得河南省医学科技创新人才工程项目和郑州大学优秀博士生培育基金资助，目前已经找到合适的磁标记条件，通过MRI可以清楚地观察到羊膜间充质干细胞的存活迁移情况。

创新要点：目前国内外关于羊膜间充质干细胞的磁标记研究尚无报道，将纳米技术与医学研究相结合、分析纳米颗粒的细胞毒性是实验的两点新颖之处。

术语解析：超顺磁性Fe3O4纳米颗粒，商品名为菲立磁，是一种负性对比剂，MRI呈现低信号，被应用于临床检测肝脏疾病及细胞的活体示踪研究。

1郑州大学第一附属医院神经外科，河南省郑州市 450052；2河南科技大学第一附属医院肿瘤科，河南省洛阳市 471003；3河南科技大学肿瘤研究所，河南省洛阳市 471003；4郑州大学生物工程系，河南省洛阳市 450001；5郑州大学基础医学院微生物与免疫学教研室，河南省洛阳市 450052；6深圳市北科细胞工程研究所，广东省深圳市 518000

王公平，男，1974年生，河南省商丘市人，主治医师，主要从事干细胞基础和临床应用方面的研究。wgpnw@126.com

通讯作者：杨波，硕士，教授，博士生导师，郑州大学第一附属医院神经外科，河南省郑州市 450052
yangbo96@126. com

河南省医学科技创新人才工程项目(2005018)*；郑州大学优秀博士生培育基金（2006008）*

摘要
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10 mL培养瓶中，密度调整为1×109 L-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20，30，40，80 mg/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1.5 mg/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12 h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12 h、36 h、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20 mg/L的Fe3O4纳米颗粒细胞标记率为60%，终浓度为30，40，80 mg/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20，30 mg/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40，80 mg/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。②30 mg/L Fe3O4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。③30 mg/L Fe3O4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。
关键词：人羊膜间充质干细胞；Fe3O4；纳米颗粒；标记

王公平，杨波，关方霞，杜英，常克亮，宋来君，胡祥，曾光伟. Fe3O4纳米化颗粒标记人羊膜间充质干细胞的合适条件[J].中国组织工程研究与临床康复，2008，12(6):1043-1046
[www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1043(ps).pdf]

Optimal condition of Fe3O4 nanoparticles labeling to human amniotic-derived mesenchymal stem cells

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 2007.①The 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.②Empirical 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 1×109 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.③Experiment 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: ①The 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.②The 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: ①Mixing PLL with Fe3O4 nanoparticles can make more Fe3O4 nanoparticles enter into cells so as to strengthen the labeling effect of cells.②The 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.③The mixture of Fe3O4 nanoparticles and PLL at the concentration of 30 mg/L is the best condition for labeling AD-MSCs.

Wang GP,Yang B, Guan FX Du Y, Chang KL, Song LJ, Hu X, Zeng GW. Optimal condition of Fe3O4 nanoparticles labeling to human amniotic-derived mesenchymal stem cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(6):1043-1046 [www.zglckf.com/zglckf/ejournal/upfiles/08-6/6k-1043(ps).pdf]

人羊膜间充质干细胞是近年新发现的一种干细胞移植来源[1]，对人羊膜间充质干细胞植入活体后存活、分化和迁移的研究，多是处死实验动物获取标本，应用病理组织学方法研究。纳米技术的兴起和发展，使活体观察人羊膜间充质干细胞的存活、分化和迁移成为可能。Fe3O4纳米化颗粒是近来发现的活体示踪剂[2-3]，标记后细胞植入体内，MRI观察细胞存活、分化和迁移情况。由于Fe3O4纳米化颗粒细胞毒性可导致标记细胞死亡[4]，寻找合适Fe3O4纳米化颗粒浓度标记细胞是活体示踪关键，本实验就其对人羊膜间充质干细胞的应用进行探讨。�

设计：随机对照细胞观察。
单位：郑州大学第一附属医院神经外科，河南科技大学第一附属医院肿瘤科。
材料：实验于2007-08在郑州大学完成。①细胞来源：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。②主要试剂和设备：Fe3O4纳米颗粒由美国泛华医药公司生产，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。倒置显微镜（OLYMPAS，IX71型，日本）。CO2培养箱（CJ-1100型，长沙长锦应用技术研究所）。
设计、实施、评估者：由第二作者进行设计与评估，干预实施为第一作者，均经过系统培训，未使用盲法评估。
方法：
人羊膜间充质干细胞的培养：自人胎盘羊膜中提取间充质干细胞[1]，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。根据细胞生长情况，每三四天更换培养液。待细胞达到80%~90%融合时，用2.5 g/L胰酶消化传代备用。
人羊膜间充质干细胞的纳米颗粒转染：取传至第3代生长旺盛的人羊膜间充质干细胞，放入含DMEM/F12培养基的10 mL培养瓶中，细胞密度1×109 L-1。设立3组：单纯Fe3O4组，又分为4个亚组，即向培养基中分别加入终浓度为20，30，40，80 mg/L的Fe3O4纳米颗粒；Fe3O4+多聚左旋赖氨酸组，又分为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1.5 mg/L多聚左旋赖氨酸；培养基对照组，仅加入DMEM/F12培养基，不含Fe3O4纳米颗粒及多聚左旋赖氨酸。各组细胞转染12 h后，均更换为DMEM/F12培养基培养3周。
Fe3O4纳米颗粒进入细胞情况普鲁士蓝染色检测：分别于标记后12 h、36 h、1周和3周，吸去培养基，磷酸盐缓冲液冲洗，40 g/L多聚甲醛固定30 min，蒸馏水冲洗3次，Perl反应液作用30 min，蒸馏水洗3次，1%核固红复染3 min，蒸馏水洗去多余的核固红，显微镜下观察Fe3O4纳米颗粒转染人羊膜间充质干细胞的情况。
细胞活性锥虫蓝染色检测：分别于标记后12 h、36 h、1周和3周，采用锥虫蓝染色间接计数活细胞数量，从而判断细胞活性。用无血清
培养基把细胞悬液稀释到200 ~2 000个/mL，在0.1 mL细胞中加入4 g /L 锥虫蓝溶液0.1 mL。轻轻混匀，数分钟后用血球计数板计数细胞。活细胞排斥锥虫蓝，因而染成蓝色的细胞即为死细胞。计算公式：细胞活性=阴性染色细胞数/100×100%。
主要观察指标：①Fe3O4纳米颗粒标记人羊膜间充质干细胞的效果。②标记后细胞活性测定。
统计学方法：由第一作者采用SPSS 12.0软件包进行统计处理，数据均以_x±s表示，组间多重比较采用LSD法，P < 0.05为差异有显著性意义。

2.1 Fe3O4纳米颗粒标记人羊膜间充质干细胞的效果
普鲁士蓝染色结果显示，终浓度为20 mg/L的Fe3O4纳米颗粒细胞标记率为60%，30，40，80 mg/L的Fe3O4纳米颗粒细胞标记率均为100%。单纯 Fe3O4组可见数量很少的蓝染Fe3O4纳米化颗粒分散于细胞浆内，见图1，Fe3O4+多聚左旋赖氨酸组可见大量蓝染Fe3O4纳米化颗粒分散于细胞浆中，部分颗粒聚集成团，见图2。培养基对照组细胞内未见蓝染颗粒。

2.2 标记后细胞活性测定标记后12 h、36 h、1周和3周锥虫蓝染色计数结果显示，与培养基对照组比较，当Fe3O4终浓度为20，30 mg/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40，80 mg/L时，上述2组细胞活性均显著降低(P < 0.05)。见表1。

干细胞磁标记是近年来影像学领域研究热点[5-10]。超顺磁性Fe3O4纳米颗粒是目前最新细胞标记物，该纳米颗粒是一种负性对比剂，MRI呈现低信号,可活体观察标记细胞生长、分化和迁移情况。有研究者成功标记人骨髓间充质干细胞[11-12]、人神经干细胞[11-14]、大鼠神经干细胞[11,15]、大鼠胚胎干细胞[16]、大鼠骨髓间充质干细胞[17]、人脐血间充质干细胞[18]、猪骨髓基质细胞[19]及鼠心肌间充质干细胞[20]等，同时研究发现，标记细胞随Fe3O4纳米化颗粒增加浓度时可出现细胞生长迟缓进而死亡，提示Fe3O4纳米化颗粒在某种浓度具有细胞毒性。因此标记细胞前，探索一个不影响细胞生长分化合适的Fe3O4纳米化颗粒浓度是活体示踪关键。
实验所用标记物为超顺磁性氧化Fe3O4纳米化颗粒，平均直径50 nm左右，颗粒表面带有负电荷。标记人羊膜间充质干细胞自人胎盘羊膜中提取，细胞表面带有负电荷，不利于Fe3O4纳米化颗粒进入。为增加Fe3O4纳米化颗粒进入细胞概率，培养液中加入转染剂多聚左旋赖氨酸，形成Fe3O4纳米颗粒与多聚左旋赖氨酸的混合物，多聚左旋赖氨酸带有正电荷，使纳米颗粒更易与细胞表面结合。研究结果表明，不加多聚左旋赖氨酸组，Fe3O4纳米化颗粒细胞标记率明显低于加入多聚左旋赖氨酸组。参照文后参考文献[4-5]，Fe3O4纳米化颗粒标记浓度本实验设置为20，30，40，80 mg/L。细胞培养12 h光镜观察见各组细胞生长良好，细胞内Fe3O4纳米化颗粒存在。Fe3O4纳米化颗粒浓度大者，细胞内进入Fe3O4纳米化颗粒较多。随后观察发现细胞分裂增殖，细胞内Fe3O4纳米化颗粒逐渐减少。普鲁士蓝染色光镜下观察，发现30，40，80 mg/L组Fe3O4纳米化颗粒细胞标记率为100%。20 mg/L组细胞转染率为60%。锥虫蓝染色细胞活性测定发现40，80 mg/L组细胞生长活性下降，死亡细胞增加，与20，30 mg/L组有显著差异。本实验结果证实Fe3O4纳米化颗粒浓度大于40 mg/L对人羊膜间充质干细胞生长造成负面影响，Fe3O4纳米化颗粒浓度小于30 mg/L对细胞生长不造成影响。实验发现Fe3O4纳米化颗粒浓度大于30 mg/L培养细胞标记率达100%，对细胞毒性随之增加，提示Fe3O4纳米化颗粒浓度30 mg/L是标记人羊膜间充质干细胞最佳浓度，多聚左旋赖氨酸浓度1.5 mg/L可以增加其进入细胞的概率，该标记条件不影响人羊膜间充质干细胞生长和分化，但是否适用于其他细胞仍需进一步研究。
本实验将Fe3O4纳米颗粒成功标记人羊膜间充质干细胞，并确定其最佳标记浓度，为进一步应用人羊膜间充质干细胞移植治疗脑损伤提供活体示踪方法奠定实验学基础。

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