CAJ | 학술논문

目的：探讨过表达多能相关转录因子Nanog对人牙髓细胞增殖及多向分化能力的影响。方法构建过表达Nanog的慢病毒载体质粒pSIN?EF2?Nanog?IRES?GFP?puro，采用psPAX和pMD2.G慢病毒包装系统转入293T细胞进行病毒液的生产和收取，转染生长良好的第3代人牙髓细胞，构建稳定过表达Nanog的人牙髓细胞株，以空载体转染的细胞为对照组，对细胞进行成脂及成牙本质诱导。采用BrdU法检测细胞的增殖情况，检测多能性转录因子Oct4和Sox2的表达，以及细胞成脂、成牙本质分化能力。采用单因素方差分析数据。结果成功构建Nanog过表达的人牙髓细胞株；BrdU法检测结果显示，Nanog过表达组比对照组细胞增殖能力强（F=90.421，P=0.000）。过表达组Oct4、Sox2 mRNA和蛋白表达水平明显高于对照组（FOct4 mRNA=71.649，POct4 mRNA=0.000；FSox2 mRNA=106.278，PSox2 mRNA=0.000；FOct4蛋白=41.632，POct4蛋白=0.002；FSox2蛋白=38.962，PSox2蛋白=0.002）。在矿化诱导条件下，Nanog过表达组DSPP、DMP1和OCN表达量、矿化结节产生量高于对照组（FDSPP=15.261， PDSPP＜0.05；FDMP1=16.235，PDMP1＜0.05；FOCN=17.019，POCN＜0.05）；成脂诱导21 d后，Nanog过表达组LPL和PPARγ2表达量及脂质产生高于对照组（FLPL=15.542，PLPL＜0.05；FPPARγ2=10.437，PPPARγ2＜0.05）。结论过表达Nanog能提高人牙髓细胞增殖能力，促进多能性转录因子Oct4、Sox2的表达，增强细胞多向分化能力。
목적：탐토과표체다능상관전록인자Nanog대인아수세포증식급다향분화능력적영향。방법구건과표체Nanog적만병독재체질립pSIN?EF2?Nanog?IRES?GFP?puro，채용psPAX화pMD2.G만병독포장계통전입293T세포진행병독액적생산화수취，전염생장량호적제3대인아수세포，구건은정과표체Nanog적인아수세포주，이공재체전염적세포위대조조，대세포진행성지급성아본질유도。채용BrdU법검측세포적증식정황，검측다능성전록인자Oct4화Sox2적표체，이급세포성지、성아본질분화능력。채용단인소방차분석수거。결과성공구건Nanog과표체적인아수세포주；BrdU법검측결과현시，Nanog과표체조비대조조세포증식능력강（F=90.421，P=0.000）。과표체조Oct4、Sox2 mRNA화단백표체수평명현고우대조조（FOct4 mRNA=71.649，POct4 mRNA=0.000；FSox2 mRNA=106.278，PSox2 mRNA=0.000；FOct4단백=41.632，POct4단백=0.002；FSox2단백=38.962，PSox2단백=0.002）。재광화유도조건하，Nanog과표체조DSPP、DMP1화OCN표체량、광화결절산생량고우대조조（FDSPP=15.261， PDSPP＜0.05；FDMP1=16.235，PDMP1＜0.05；FOCN=17.019，POCN＜0.05）；성지유도21 d후，Nanog과표체조LPL화PPARγ2표체량급지질산생고우대조조（FLPL=15.542，PLPL＜0.05；FPPARγ2=10.437，PPPARγ2＜0.05）。결론과표체Nanog능제고인아수세포증식능력，촉진다능성전록인자Oct4、Sox2적표체，증강세포다향분화능력。
Objective To investigate the effect of overexpression Nanog on the cell proliferationand multipotent differentiation potentiality of human dental pulp cells (hDPCs). Methods Primary human pulp cells were transfected with plasmid pSIN?EF2?Nanog?IRES?GFP?puro by lentiviral transfection and stable nanog?expressing cell line was screened under fluorescence microscope. The effect of Nanog on the proliferation of hDPCs was examined with BrdU assay. The expressions levels of pluripotency factor Oct 4 and Sox2 were detected using real?time PCR and western blotting. Nanog?hDPCs were cultured in odontogenic and adipogenic induction media respectively. The odontogenic and adipogenic differentiation potentiality were evaluated by Alizarin red S staining and Oil red O staining,and the odontogenic and adipogenic markers were detected using real?time PCR. Results The recombinant lentiviral vector containing Nanog was successfully constructed and showed high efficiency during infection in hDPCs. The BrdU assay showed that the growth rate of Nanog?hDPCs was higher compared with the controls (F =90.421,P=0.000). The expressions of Oct4 and Sox2 were increased in Nanog?hDPCs in comparison with the controls(FOct4 mRNA=71.649,POct4 mRNA=0.000;FSox2 mRNA=106.278,PSox2 mRNA=0.000;FOct4=41.632,POct4=0.002;FSox2 = 38.962,PSox2 = 0.002). Furthermore,the mRNA levels of DSPP,DMP1,and OCN were increased,and the number of mineralized nodules was significantly higher in Nanog?hDPCs (FDSPP =15.261,PDSPP < 0.05;FDMP1 = 16.235,PDMP1 < 0.05;FOCN = 17.019,POCN < 0.05). Meanwhile,significantly higher mRNA expression levels of LPL and PPARγ2 and more lipid droplets were observed in Nanog?hDPCs compared to the controls(FLPL=15.542,PLPL<0.05;FPPARγ2=10.437,PPPARγ2<0.05). Conclusion Nanog overexpression enhanced cell proliferation,upregulated the expression of pluripotent markers,and promoted the multipotent differentiation potential(odontogenic and adipogenic differentiation)of hDPCs.