新型超顺磁性氧化铁标记SD大鼠脂肪干细胞的有效性及安全性研究

目的：采用新型超顺磁性氧化铁对 SD 大鼠来源的脂肪干细胞（ADSCs）进行标记，并与既往商用 SPIO 标记效果进行对比，探讨这种新型超顺磁性氧化铁标记的有效性及安全性。方法：分离、纯化、鉴定 SD 大鼠来源的 ADSCs，然后分不同浓度组（0、6、12、25、50和100μg／mL）和时间组（6、12、24和48 h）进行标记，通过普鲁士蓝染色测定铁标记率；在不影响细胞形态的前提下，对达到95％以上铁染色率的孵育浓度、时间进行标记安全性检测，包括活力、增殖力、细胞表面抗原表达；采用透射电子显微镜观察标记细胞的超微结构，采用 ICP-AES 对标记细胞内的铁含量进行测定，并与商用SPIO 标记效果进行对比。结果：在无细胞毒性的前提下，新型 SPIO 达到95％以上铁染色率的孵育浓度是12和25μg／mL，孵育时间是12 h；ICP-AES 检测显示具有表面正电荷的聚乙二醇（PEG）／聚乙烯亚胺（PEI）修饰的 SPIO 标记后细胞内的铁含量达到35．4 pg／cell（25μg／mL 中孵育12h 后）和20．16 pg／cell（12μg／mL 中孵育12h 后），并随着孵育浓度的增加，细胞内的铁含量增加；而具有表面零电荷的 PEG／聚乙烯吡咯烷酮（PVP）修饰的 SPIO 标记后的铁含量仅为6．96 pg／cell（25μg／mL 中孵育12h 后）；透射电子显微镜显示标记后细胞器结构完整，内吸收的 SPIO 主要位于细胞质内的囊泡和溶酶体中。结论：新型 SPIO 在适当孵育浓度和时间下可以安全、快速标记 ADSCs；PEG／PEI 修饰的 SPIO 标记效果要远远比既往商用的 SPIO 快速有效，可作为一种优势的新型磁性标记物用于干细胞标记；而 PEG／PVP 修饰的SPIO 比起既往商用的 SPIO 并无明显优势，说明表面电荷在细胞标记中占有极其重要的角色。

The research of effectiveness and safety on SD rat adipose-derived stem cells labeled with a new type of superparamagnetic iron ;oxide nanoparticles

Objective:The aim of the present study was to explore the efficacy and safety of a new type of superpara-magnetic nanoparticles (SPION)by labeling the SD rat adipose-derived stem cells (ADSCs)with SPION and comparing with our previous research results on commercial superparamagnetic iron oxide (SPIO).Methods:First,the SD rat ADSCs were incubated and labeled by different concentration groups (0,6,12,25,50,100μg/mL,respectively)and by different time groups (6,12,24,48h,respectively)after isolation,purification and identification.The labeling rate of iron oxide nanoparti-cles were detected by prussian blue staining.Second,the safety of high labeling efficacy (>95%)in ADSCs were evaluated, under the condition of not affecting the cell morphology,with viability,proliferation,surface makers identification and neuro-nal induction.Third,the ultrastructure of labeled ADSCs were oberved by transmission electron microscopy (TEM). Fourth,the iron content of labeled ADSCs were measured with ICP-AES and compared with our previous research results on commercial SPIO.Results:Under the premise of with no cytotoxicity,the incubation concentration for high labeling effi-ciency (>95%)of the new SPIO were 12 and 25μg/mL at the incubation time of 12h.The ICP-AES measurements showed that the iron content in ADSCs was 35.4pg/cell or 20.16pg/cell at the incubation of PEG/PEI SPIO which carried perma-nent positive surface charge,whereas only 6.96pg/cell at the incubation of PEG/PVP SPIO (25μg/mL,12h).TEM ima-ging showed that the cellular ultrastructure was intact,and the nanoparticles uptake was dominantly located in the vesicles and lysosomes.Conclusion:At the incubation of appropriate concentration and time,ADSCs can be safely and quickly labeled with new type of paramagnetic nanoparticles.Compared with the commercial SPIO,the new type of PEG/PEI SPIO trends to be a more advantaged magnetic tracer to label stem cells.In contrast,the advantage of PEG/PVP SPIO is not apparent, which indicates that surface charge have played an extremely important role in stem cell magnetic labeling.