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

摘要/Abstract

摘要： 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；CCK-8结果显示，第1天50、100?μg/mL浓度下的细胞活性明显升高，而浓度达到300?μg/mL时，细胞活性降低，但细胞相对存活率仍高于80%；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，且在共培养9?d后仍能看到荧光；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。

关键词:

碳点, 骨髓间充质干细胞, 示踪, 成骨分化

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

史凡黄鉴栎刘梅王丹章非敏. 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[J]. 口腔生物医学, 2021, 12(1): 5-11.

参考文献 31

[1]	Ng KS, Kuncewicz TM, Karp JM.Beyond hit-and-run: Stem cells leave a lasting memory[J].Cell Metab, 2015, 22(4):541-543
[2]	Ankrum JA, Ong JF, Karp JM.Mesenchymal stem cells: Immune evasive, not immune privileged[J].Nat Biotechnol, 2014, 32(3):252-260
[3]	Fu X, Liu G, Halim A, et al.Mesenchymal Stem Cell Migration and Tissue Repair[J].Cells, 2019, 8(8):784-
[4]	Shao D, Lu M, Xu D, et al.Carbon dots for tracking and promoting the osteogenic differentiation of mesenchymal stem cells[J].Biomater Sci, 2017, 5(9):1820-1827
[5]	Yang X, Wang Y, Shen X, et al.One-step synthesis of photoluminescent carbon dots with excitation-independent emission for selective bioimaging and gene delivery[J].J Colloid Interface Sci, 2017, 492:1-7
[6]	Parvin N, Mandal TK.Dually emissive P,N-co-doped carbon dots for fluorescent and photoacoustic tissue imaging in living mice[J].Microchim Acta, 2017, 184(4):1117-1125
[7]	Boakye-Yiadom KO, Kesse S, Opoku-Damoah Y, et al.Carbon dots: Applications in bioimaging and theranostics[J].Int J Pharm, 2019, 564:308-317
[8]	Du J, Xu N, Fan J, et al.Carbon dots for in vivo bioimaging and theranostics[J].Small, 2019, 15(32):e1805087-
[9]	Mondal S, Yucknovsky A, Akulov K, et al.Efficient Photosensitizing Capabilities and Ultrafast Carrier Dynamics of Doped Carbon Dots[J].J Am Chem Soc, 2019, 141(38):15413-15422
[10]	Su R, Wang D, Liu M, et al.Subgram-Scale Synthesis of Biomass Waste-Derived Fluorescent Carbon Dots in Subcritical Water for Bioimaging, Sensing, and Solid-State Patterning[J].ACS Omega, 2018, 3(10):13211-13218
[11]	Zhang M, Zhao X, Fang Z, et al.Fabrication of HA/PEI-functionalized carbon dots for tumor targeting, intracellular imaging and gene delivery[J].RSC Adv, 2017, 7(6):3369-3375
[12]	Wang D, Wang Z, Zhan Q, et al.Facile and scalable preparation of fluorescent carbon dots for multifunctional applications[J].Engineering, 2017, 3(3):402-408
[13]	Callera F, de Melo CMTP.Magnetic resonance tracking of magnetically labeled autologous bone marrow CD34+ cells transplanted into the spinal cord via lumbar puncture technique in patients with chronic spinal cord injury: CD34+ cells' migration into the injured site[J].Stem Cells Dev, 2007, 16(3):461-466
[14]	Bartunek J, Vanderheyden M, Vandekerckhove B, et al.Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: Feasibility and safety[J].Circulation, 2005, 112(9 Suppl):I178-I183
[15]	Strauer BE, Brehm M, Zeus T, et al.Regeneration of human infarcted heart muscle by intracoronary autologous bone marrow cell transplantation in chronic coronary artery disease: The IACT study[J].J Am Coll Cardiol, 2005, 46(9):1651-1658
[16]	Teston E, Maldiney T, Marangon I, et al.Nanohybrids with magnetic and persistent luminescence properties for cell labeling,tracking,in vivo real-time imaging,and magnetic vectorization[J].Small, 2018, 14(16):e1800020-
[17]	Li J, Lee WY, Wu TY, et al.Multifunctional quantum dot nanoparticles for effective differentiation and long-term tracking of human mesenchymal stem cells in vitro and in vivo[J].Adv Healthcare Mater, 2016, 5(9):1049-1057
[18]	Serganova I, Blasberg RG.Molecular imaging with reporter genes: Has its promise been delivered?[J].J Nucl Med, 2019, 60(12):1665-1681
[19]	Govindan S, Oberst P, Jabaudon D.In vivo pulse labeling of isochronic cohorts of cells in the central nervous system using FlashTag[J].Nat Protoc, 2018, 13(10):2297-2311
[20]	Ji F, Duan HG, Zheng CQ, et al.Comparison of chloromethyl-dialkylcarbocyanine and green fluorescent protein for labeling human umbilical mesenchymal stem cells[J].Biotechnol Lett, 2015, 37(2):437-447
[21]	Kang YF, Fang YW, Li YH, et al.Nucleus-staining with biomolecule-mimicking nitrogen-doped carbon dots prepared by a fast neutralization heat strategy[J].Chem Commun, 2015, 51(95):16956-16959
[22]	Rong M, Feng YF, Wang Y, et al.One-pot solid phase pyrolysis synthesis of nitrogen-doped carbon dots for Fe3+ sensing and bioimaging[J].Sensor Actuat B: Chem, 2017, 245:868-874
[23]	Li HT, Liu R, Kong W, et al.Carbon quantum dots with photo-generated proton property as efficient visible light controlled acid catalyst[J].Nanoscale, 2014, 6(2):867-873
[24]	Pan L, Sun S, Zhang A, et al.Truly fluorescent excitation-dependent carbon dots and their applications in multicolor cellular imaging and multidimensional sensing[J].Adv Mater, 2015, 27(47):7782-7787
[25]	Cao L, Wang X, Meziani MJ, et al.Carbon dots for multiphoton bioimaging[J].J Am Chem Soc, 2007, 129(37):11318-11319
[26]	Han Y, Zhang F, Zhang J, et al.Bioactive carbon dots direct the osteogenic differentiation of human bone marrow mesenchymal stem cells[J].Colloids Surf B Biointerfaces, 2019, 179:1-8
[27]	Ding H, Wei JS, Zhong N, et al.Highly efficient red-emitting carbon dots with gram-scale yield for bioimaging[J].Langmuir, 2017, 33(44):12635-12642
[28]	Tao H, Yang K, Ma Z, et al.In vivo NIR fluorescence imaging,biodistribution,and toxicology of photoluminescent carbon dots produced from carbon nanotubes and graphite[J].Small, 2012, 8(2):281-290
[29]	Li D, Jing P, Sun L, et al.Near-Infrared Excitation/Emission and Multiphoton-Induced Fluorescence of Carbon Dots[J].Adv Mater, 2018, 30(13):e1705913-
[30]	Ding H, Wei JS, Zhang P, et al.Solvent-controlled synthesis of highly luminescent carbon dots with a wide color gamut and narrowed emission peak widths[J].Small, 2018, 14(22):e1800612-
[31]	Ko HY, Chang YW, Paramasivam G, et al.In vivo imaging of tumour bearing near-infrared fluorescence-emitting carbon nanodots derived from tire soot[J].Chem Commun, 2013, 49(87):10290-