1950 MHz射频电磁场对人脐带间充质干细胞增殖和成骨分化的影响研究

Effects of 1950 MHz Radiofrequency Electromagnetic Fields on the Proliferation and Osteogenic Differentiation of Human Umbilical Cord Mesenchymal Stem Cells

目的:间充质干细胞(Mesenchymal stem cells,MSCs)具有广阔的临床应用前景,但由于其体外增殖和定向分化等问题,制约了其进一步应用。本研究拟探讨1950MHz射频电磁场(Radio-frequency electromagnetic fields,RF-EMF)对人脐带间充质干细胞(Human umbilical cord mesenchymal stem cells,hUC-MSCs)增殖和成骨方向分化的影响,以期为MSCs的体外增殖和定向分化提供一条新途径。方法:华通氏胶组织块法分离培养人脐带间充质干细胞,流式细胞仪检测间充质干细胞特异性标志物。选择鉴定后的第3至第6代(P3-P6)hUC-MSCs用于实验。将hUC-MSCs细胞暴露或假暴露于频率为1950 MHz,比吸收率(Specific absorption rate,SAR)分别为0.5,1.0和2.0 W/kg的RF-EMF中,每天暴露1 h(5 min开,10 min关),连续暴露7 d。暴露结束后,流式细胞仪检测细胞周期,免疫荧光检测增殖相关蛋白Ki67表达,连续6天用CCK-8方法检测细胞数。在成骨分化研究中,将P3代的hUC-MSCs随机分为假暴露(sham)组,射频辐射暴露(RF)组,成骨诱导培养基组(Induction medium,OM)和成骨诱导培养基联合射频辐射暴露(OM+RF)组,暴露SAR值为2.0 W/kg,其它参数不变。暴露结束后立即检测细胞的碱性磷酸酶(Alkaline phosphatase,ALP)活性。结果:原代培养的细胞具有MSC典型外观,且表达MSCs特异性表面抗原。与sham组相比,不同SAR值RF暴露后,hUC-MSCs的增殖能力无明显变化,S期细胞比例及Ki67蛋白水平也无显著改变。此外,hUC-MSCs经SAR值为2.0W/kg的RF暴露7 d,与sham组相比其ALP活性无显著变化。与OM组相比,OM+RF组的ALP活性亦无显著改变。结论:华通氏胶组织块法能够培养出纯度较高的间充质干细胞,本实验条件下的1950 MHz射频电磁场对hUC-MSCs的增殖和成骨分化均无显著影响。

Objective： Mesenchymal stem cells（MSCs） has wide prospect of clinical application, but its proliferation and directional differentiation in vitro restrict its further application. In the study, we explored the effects of 1950 MHz radiofrequency electromagnetic fields（RF-EMF） on the proliferation and osteogenic differentiation of human umbilical cord mesenchymal stem cells（hUC-MSCs）,in order to provide a new way for the proliferation and differentiation of MSCs in vitro. Methods： The hUC-MSCs were cultured by explant method of Wharton＇s jelly and the specific markers of mesenchymal stem cells were detected by flow cytometry（FCM）. The hUC-MSCs of passage 3 to 6 were assigned and intermittent exposed or sham-exposed to 1950 MHz RF-EMF at specific absorption rates（SARs） of 0.5, 1.0 or 2.0 W/kg for 7 consecutive days（1 h/d）, 5 min on and 10 min off. After exposure, cell viability was detected by CCK-8, cell cycle was measured by flow cytometry, and Ki67 protein expression was tested by immunofluorescence. In the study of osteogenic differentiation, the hUC-MSCs of passage 3 were divided into sham group, RF group, induction medium（OM） group and OM＋RF group. After the same exposure above, except the SAR is 2.0 W/kg, the alkaline phosphatase（ALP） activity was measured immediately. Results： The cells cultured by explant method showed typical appearance of MSCs, and expressed MSCs specific surface antigen positively. Compared with sham-exposed cells, the proliferation capacity of hUC-MSCs did not change significantly after RF-EMF exposure at different SAR values, and the percentage of cells in S phase and the level of Ki67 protein expression also did not change obviously.After exposure, the activity of ALP between sham group and RF group, as well as OM and OM＋RF group, had no significant differences. Conclusions： The hUC-MSCs cells could be cultured by the method of Wharton＇s jelly explant with high purity. 1950 MHz RF-EMF in the experiment had no significant effects on the proliferation and osteogenic differentiation of hUC-MSCs in vitro.