碳点示踪并促进大鼠骨髓间充质干细胞成骨分化的初步研究

Abstract

Abstract: Objective:To investigate the possibility of using long-wave carbon dots to trace rat bone marrow mesenchymal stem cells (BMSCs) and its effects on cell activity and osteogenic differentiation. Methods:CDs were characterized by TEM and FT-IR. CCK8 assay and flow cytometry were applied to screen the optimal concentration of CDs for imaging. After the CDs were co-cultured with BMSCs, the pathways of cells up taking CDs were investigated through a variety of transmembrane inhibitors and culturing at 4 ℃ low temperature. laser scanning confocal microscopy was used to trace the labeled cells for 9 days. Lysosome fluorescent probe was used to observe the distribution of CDs. ALP activity was measured at 4 days and 7 days, mineralization ability was detected by alizarin red staining at 21 days, and the osteogenic markers Runx2, Ocn, Opn, Bmp2 and Osterix were detected by PCR at 7 days and 14 days. Results:TEM showed that CDs were spherical particles and the average particle size was 2.17 mm. Ft-IR analysis showed that the surface of CDs was rich in hydroxyl and amino groups. CCK-8 results showed that the cell activity increased significantly when the concentration of CDs was 50 and 100 μg/mL at the 1st day. When the concentration reached 300 μg/mL, the cell activity decreased, but was still higher than 80%. Flow cytometry results showed that the labeling efficiency increased with the increase of CDs concentration. The red fluorescence of CDs overlapped partially with the green fluorescence of lysosomes and partially located in the cytoplasm, but did not enter the nucleus. The fluorescence could still be seen after 9 days. When the energy-dependent endocytosis pathway was inhibited at 4 ℃, the uptake amounts of CDs in the cells were significantly decreased. Compared with the control group, the ALP activity, mineralization ability and the expressions of Runx2, Ocn, Opn, Bmp2, Osterix were all increased in the CDs group. Conclusions:CDs have the potential to trace BMSCs and regulate the osteogenic differentiation.

Key words: carbon dots, bone marrow mesenchymal stem cells, trace, osteogenic differentiation

References 31

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The preliminary study of CDs for tracing and promoting osteogenic differentiation of rat BMSCs

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