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

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

目的:利用长波长碳点(CDs)标记大鼠骨髓间充质干细胞(BMSCs),探讨其示踪可能性及对细胞成骨分化的影响。方法:用透射电镜(TEM)、傅立叶红外光谱仪(FT-IR)检测CDs表征;用CCK-8法和流式细胞术筛选出CDs最佳成像浓度,将最佳浓度的CDs与BMSCs共培养后,通过多种跨膜抑制剂及4℃低温条件,探讨细胞摄取碳点的途径,并利用共聚焦显微镜观察细胞的成像特点,通过溶酶体探针进行荧光共定位以观察CDs在细胞内的分布,同时进行9 d的示踪;在共培养的第4、7天测细胞的碱性磷酸酶(ALP)活性,第21天通过茜素红检测矿化能力,第7和14天通过实时定量RT-PCR检测Runt相关转录因子2(Runx2)、骨钙素(Ocn)、骨桥蛋白(Opn)、骨形态发生蛋白2(Bmp2)、成骨细胞特异性转录因子Osterix等成骨相关基因的表达。结果:TEM下见CDs为球形颗粒,粒径平均为2.17 mm;FT-IR分析CDs表面富含羟基、氨基等基团;第1天50、100μg/m L浓度下的细胞活性明显升高,而浓度达到300μg/m L时,细胞活性降低,流式细胞术结果显示随CDs浓度升高,BMSCs的标记效率升高;成像结果显示BMSCs呈红色荧光图像,荧光区部分与溶酶体绿色荧光重叠,部分位于细胞质,但不进入细胞核;4℃培养抑制能量依赖性的内吞途径时,细胞摄取CDs的量明显减少;与对照组相比,CDs组的ALP活性、矿化能力及成骨相关基因表达均有升高。结论:CDs具有示踪并调节BMSCs成骨分化功能的潜力。

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. The cell activity increased significantly when the concentration of CDs was 50 and 100μg/m L at the 1 st day. When the concentration reached 300 μg/m L,the cell activity decreased. 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. 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.