Effectene介导钆喷酸葡胺标记人脐带间充质干细胞及体外磁共振成像研究

Study on human umbilical cord mesenchymal stem cells labeled by Effectene mediated gadopentetate dimeglumine and their MRI imaging in vitro

目的应用Effectene介导钆喷酸葡胺(Gd-DTPA)标记人脐带间充质干细胞(hUCMSCs),研究磁标记干细胞的生物学特性,探讨Gd-DTPA标记干细胞体外磁共振成像(MRI)规律。方法组织块贴壁法分离纯化hUCMSCs,通过传代培养、扩增,鉴定细胞在体外的生物学特性。应用Effectene转染Gd-DTPA标记hUCMSCs,计数法检测Gd-DTPA标记干细胞的增殖能力,体外诱导其向成脂细胞和成骨细胞分化,观察Gd-DTPA影响hUCMSCs的生物学特性。应用1.5T临床应用型MRI系统,观察Gd-DTPA标记hUCMSCs的信号强度随细胞传代的变化规律,并探索MRI的最低细胞量。结果应用组织块贴壁法接种2周后获得原代细胞,细胞呈长梭形,漩涡样生长,传2代后细胞形态更为均匀一致。流式细胞仪检测第3代细胞高表达CD29、CD44、CD90和CD105,不表达CD31、CD45、CD40和HLA-DR。体外定向诱导能分化为成脂细胞和成骨细胞。Effectene能成功转染Gd-DTPA进入干细胞,检测标记后的干细胞增殖能力未受到影响,并能在体外诱导向成骨细胞和成脂细胞分化。体外MRI扫描Gd-DTPA标记的干细胞在T1WI呈现高信号,体外持续示踪时间约12 d。结论应用组织块贴壁法能有效分离纯化hUCMSCs。应用Effectene转染Gd-DTPA标记hUCMSCs进行体外MRI示踪是可行的。

Objective To apply the human umbilical cord mesenchymal stem cells （hUCMSCs） labeled by Effectene mediated gadopentetate dimeglumine （Gd-DTPA） and to investigate the biologic characteristics of magnetically labeled stem cells and the rules of MRI of Gd-DTPA labeled MSCs in vitro. Methods The hUCMSCs was separated and purified by the tissue adherent method. The biological characteristics of cells in vitro were identified after being subcultured and amplified. The hUCMSCs was marked by Effectene transfected Gd-DTPA. The proliferation ability of Gd-DTPA labeled stem cells was detected by the counting method. Then stem cells were induced to differentiate to adipocytes and osteoblasts in vitro. The effects of Gd- DTPA on the biological characteristics of hUCMSCs were observed. The clinical 1.5T MRI system was used to observe changes of the signal intensity of Gd-DTPA labeled hUCMSCs with cell passages and explore the minimum cell mass of MRI. Results Primary cells were obtained by applying the tissue adherent method for two weeks. The cells were fusiform with whirlpool-like growth. After two passages, cells were morphologically homogeneous. The flow cytometry detected that the third passage cells had high expressions of CD29, CD44, CD90, and CD105 and no expression of CD31, CD40, CD45, and HLA-DR. The cells could be induced toadipocytes and osteoblasts in vitro. Effectene could successfully transfected with Gd-DTPA into stem cells. The proliferation of labeled stem cells was not affected and the cells could be induced to differentiate towards osteoblast or adipocyte in vitro. The results of MRI scanning of Gd-DTPA labeled stem cells showed high signal on T1WI and the continuous tracing lasted about 12 d in vitro. Conclusion The tissue adherent method can effectively isolate and purify hUCMSCs. It is feasible to perform MRI tracking in vitro through hUCMSCs labeled by Effectene transfected Gd-DTPA.