葡萄糖酸锌碳点在细胞成像和体外促进成骨分化中的作用
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摘要
目的:通过一步水热法合成葡萄糖酸锌碳点(Zn-CDs),探讨Zn-CDs的细胞成像及其对小鼠前成骨细胞向成骨方向分化的促进作用。方法:一步水热法合成Zn-CDs,采用透射电子显微镜(TEM)、荧光光谱仪和傅里叶变换红外光谱仪(FT-IR)观察检测Zn-CDs的表征。将不同浓度(0.01、0.10、1.00、10.00、100.00和1 000.00mg·L~(-1))Zn-CDs浸提液与小鼠前成骨细胞系MC3T3-E1共培养作为实验组,空白对照组仅加入细胞培养液。采用MTT法检测各组MC3T-E1细胞相对增殖率(RGR);采用激光共聚焦成像观察MC3T3-E1细胞的成像特点;采用qRT-PCR法检测各组MC3T3-E1细胞中Runt相关转录因子2基因(Runx2)、碱性磷酸酶基因(ALP)和骨钙素(OC)mRNA相对表达水平;茜素红染色检测各组细胞中钙化结节数。结果:TEM检测,成功合成粒径为5.25nm的Zn-CDs。荧光光谱,Zn-CDs具有360nm紫外光激发和450nm蓝光发射的荧光性质,并表现激发波长依赖特性。FT-IR检测,Zn-CDs表面主要由羧基和羟基基团构成。与空白对照组比较,共培养24h时1 000.00mg·L~(-1)Zn-CDs组MC3T3-E1细胞的RGR明显降低(P<0.01)。荧光成像,Zn-CDs与MC3T3-E1细胞共培养后,细胞呈现蓝色、绿色和红色的荧光图像,形态轮廓清楚且荧光强度细胞质比细胞核强。qRT-PCR检测,随着Zn-CDs浓度的增加,MC3T3-E1细胞中Runx2、ALP和OCmRNA相对表达水平逐渐升高。茜素红染色,诱导21d后不同浓度Zn-CDs组MC3T3-E1细胞中钙结节数多于空白对照组。结论:Zn-CDs可以有效地进行MC3T3-E1细胞荧光成像,且Zn-CDs具有一定的促MC3T3-E1细胞向成骨方向分化的作用。
Objective:To synthesize the zinc gluconate carbon dots(Zn-CDs)by one-step hydrothermal method,and to investigate their effects on the cell imaging and inducing osteoblastic differentiation of preosteoblasts in the mice.Methods:The Zn-CDs were synthesized by one-step hydrothermal method,and the characteristics were observed and detected by transmission electron microscope(TEM),Fourier transform-infrared spectrum(FT-IR)and fluorescence spectrometer.The MC3T3-E1 cells were divided into blank control group and experimental groups;different concentrations(0.01,0.10,1.00,10.00,100.00,1 000 mg·L~(-1))of Zn-CDs were added into the cells in experimental groups,and nothing was added into the cells in blank control group.MTT assay was used to determin the relative growth rate(RGR)of MC3T3-E1 cells in various groups;the imaging characteristics of MC3T3-E1 cells were observed under confocal microscope;the relative expression levels of Runt-relateed transcription factor-2(Runx2),alkaline phosphatase(ALP)and osteocalcin(OC)mRNA in the MC3T3-E1 cells in various groups were detected by qRT-PCR;the number of calcified nodules was detected by alizarin red staining.Results:The TEM results showed that the particle size of Zn-CDs was about 5.25 nm.The fluorescence spectrometer results showed that the Zn-CDs had the fluorescence properties of 360 nm ultraviolet excited light and 450 nm blue emission light,and the Zn-CDs showed the characteristics of excitation wavelength dependence.The FT-IR results showed that the surface of Zn-CDs was mainly composed of carboxyl groups and hydroxyl groups.Compared with blank control group,the RGR of MC T3-E1 cells in 1 000.00 mg·L~(-1) Zn-CDs group was decreased significantly at 24 hafter co-culture(P<0.01).The results of fluorescence imaging showed that the blue,green and red fluorescence in the MC3T3-E1 cells,the outline was clear,and the fluorescence intensity of the cytoplasm was stronger than that of the nucleus after co-cultured with Zn-CDs.The qRT-PCR results showed that the relative expression levels of Runx2,ALP and OC mRNA were significantly increased with the increasing of ZnCDs concentration.The alizarin red staining results showed that the number of calcium deposits in the MC3T3-E1 cells in different concentrations of Zn-CDs groups were more than that in blank control group after induced for 21 d.Conclusion:Zn-CDs can effectively perform the fluorescence imaging in the MC3 T3-E1 cells,and Zn-CDs have a certain ability to promote the osteoblastic differentiation of the MC3T3-E1 cells.
Objective:To synthesize the zinc gluconate carbon dots(Zn-CDs)by one-step hydrothermal method,and to investigate their effects on the cell imaging and inducing osteoblastic differentiation of preosteoblasts in the mice.Methods:The Zn-CDs were synthesized by one-step hydrothermal method,and the characteristics were observed and detected by transmission electron microscope(TEM),Fourier transform-infrared spectrum(FT-IR)and fluorescence spectrometer.The MC3T3-E1 cells were divided into blank control group and experimental groups;different concentrations(0.01,0.10,1.00,10.00,100.00,1 000 mg·L~(-1))of Zn-CDs were added into the cells in experimental groups,and nothing was added into the cells in blank control group.MTT assay was used to determin the relative growth rate(RGR)of MC3T3-E1 cells in various groups;the imaging characteristics of MC3T3-E1 cells were observed under confocal microscope;the relative expression levels of Runt-relateed transcription factor-2(Runx2),alkaline phosphatase(ALP)and osteocalcin(OC)mRNA in the MC3T3-E1 cells in various groups were detected by qRT-PCR;the number of calcified nodules was detected by alizarin red staining.Results:The TEM results showed that the particle size of Zn-CDs was about 5.25 nm.The fluorescence spectrometer results showed that the Zn-CDs had the fluorescence properties of 360 nm ultraviolet excited light and 450 nm blue emission light,and the Zn-CDs showed the characteristics of excitation wavelength dependence.The FT-IR results showed that the surface of Zn-CDs was mainly composed of carboxyl groups and hydroxyl groups.Compared with blank control group,the RGR of MC T3-E1 cells in 1 000.00 mg·L~(-1) Zn-CDs group was decreased significantly at 24 hafter co-culture(P<0.01).The results of fluorescence imaging showed that the blue,green and red fluorescence in the MC3T3-E1 cells,the outline was clear,and the fluorescence intensity of the cytoplasm was stronger than that of the nucleus after co-cultured with Zn-CDs.The qRT-PCR results showed that the relative expression levels of Runx2,ALP and OC mRNA were significantly increased with the increasing of ZnCDs concentration.The alizarin red staining results showed that the number of calcium deposits in the MC3T3-E1 cells in different concentrations of Zn-CDs groups were more than that in blank control group after induced for 21 d.Conclusion:Zn-CDs can effectively perform the fluorescence imaging in the MC3 T3-E1 cells,and Zn-CDs have a certain ability to promote the osteoblastic differentiation of the MC3T3-E1 cells.
引文
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[17]YANG J W,JEONG B C,PARK J,et al.PHF20positively regulates osteoblast differentiation via increasing the texpression and activation of runx2 with enrichment of H3K4me3[J].Sci Rep,2017,7(1):8060.
[18]KOMORI T.Roles of runx2in skeletal development[J].Oxygen Transport Tissue,2017,962(1):83-93.
[19]HE Y F,MA Y,GAO C,et al.Iron overload inhibits osteoblast biological activity through oxidative stress[J].Biol Trace Elem Res,2013,152(2):292-296.
[20]POUNDARIK A A,BOSKEY A,GUNDBERG C,et al.Biomolecular regulation,composition and nanoarchitecture of bone mineral[J].Sci Rep,2018,8(1):1191.
[21]晋瑞,李嫚,雷喆,等.转化生长因子-β1对MC3T3-E1小鼠成骨细胞骨形成的影响[J].郑州大学学报:医学版,2018,53(3):378-381.
[2]HIENZ S A,PALIWAL S,IVANOVSKI S.Mechanisms of bone resorption in periodontitis[J].J Immunol Res,2015,2015(1):615486.
[3]李格格,唐秋玲,潘佳慧,等.PMX205抗炎作用的体外研究[J].口腔医学研究,2017,33(12):1246-1249.
[4]GAN K,LIU H,JIANG L,et al.Bioactivity and antibacterial effect of nitrogen plasma immersion ion implantation on polyetheretherketone[J].Dent Mater,2016,32(11):e263-e274.
[5]SUN Y P,WANG P,LU Z M,et al.Host-guest carbon dots for enhanced optical properties and beyond[J].Sci Rep,2015,5(1):12354.
[6]SUN Y P,ZHOU B,LIN Y,et al.Quantum-sized carbon dots for bright and colorful photoluminescence[J].J Am Chem Soc,2006,128(24):7756-7757.
[7]SUN C,ZHANG Y,WANG P,et al.Synthesis of nitrogen and sulfur co-doped carbon dots from garlic for selective detection of Fe3+[J].Nanoscale Res Lett,2016,11(1):110.
[8]LI H,KANG Z,LIU Y,et al.Carbon nanodots:synthesis,properties and applications[J].J Mater Chem,2012,22(46):24230-24253.
[9]DING C Q,ZHU A W,TIAN Y.Functional surface engineering of C-dots for fluorescent biosensing and in vivo bioimaging[J].Accounts Chem Res,2014,47(1):20.
[10]FU C,YANG X,TAN S,et al.Enhancing cell proliferation and osteogenic differentiation of MC3T3-E1pre-osteoblasts by BMP-2 Delivery in graphene oxide-incorporated PLGA/HAbiodegradable microcarriers[J].Sci Rep,2017,7(1):12549.
[11]VASIMALAI N,VILAS-BOAS V,GALLO J,et al.Green synthesis of fluorescent carbon dots from spices for in vitro imaging and tumour cell growth inhibition[J].Beilstein JNanotechnol,2018,9:530-544.
[12]LUO P G,SAHU S,YANG S T,et al.Carbon“quantum”dots for optical bioimaging[J].J Mater Chem B,2013,1(16):2116-2127.
[13]ATABAEV T S.Doped carbon dots for sensing and bioimaging applications:a minireview[J].Nanomaterials(Basel),2018,8(5):342.
[14]LUO P G,YANG F,YANG S T,et al.Carbon-based quantum dots for fluorescence imaging of cells and tissues[J].Rsc Adv,2014,4(21):10791-10807.
[15]YANG S T,WANG X,WANG H,et al.Carbon dots as nontoxic and high-performance fluorescence imaging agents[J].J Phys Chem C Nanomater Interfaces,2009,113(42):18110.
[16]ZHANG F,REN L F,LIN H S,et al.The optimal dose of recombinant human osteogenic protein-1 enhances differentiation of mouse osteoblast-like cells:an in vitro study[J].Arch Oral Biol,2012,57(5):460-468.
[17]YANG J W,JEONG B C,PARK J,et al.PHF20positively regulates osteoblast differentiation via increasing the texpression and activation of runx2 with enrichment of H3K4me3[J].Sci Rep,2017,7(1):8060.
[18]KOMORI T.Roles of runx2in skeletal development[J].Oxygen Transport Tissue,2017,962(1):83-93.
[19]HE Y F,MA Y,GAO C,et al.Iron overload inhibits osteoblast biological activity through oxidative stress[J].Biol Trace Elem Res,2013,152(2):292-296.
[20]POUNDARIK A A,BOSKEY A,GUNDBERG C,et al.Biomolecular regulation,composition and nanoarchitecture of bone mineral[J].Sci Rep,2018,8(1):1191.
[21]晋瑞,李嫚,雷喆,等.转化生长因子-β1对MC3T3-E1小鼠成骨细胞骨形成的影响[J].郑州大学学报:医学版,2018,53(3):378-381.