探讨p53在低氧抑制人牙周膜成纤维细胞（PDLCs）成骨分化中的作用。

体外正常氧（20% O2）和低氧（1% O2）中培养PDLCs细胞48 h，Western免疫印迹检测12、24与48 h时p53与HIF-1α的表达水平；小干扰RNA（Si-HIF1α）转染PDLCs，验证敲低HIF-1α表达后p53水平变化；进一步通过小干扰RNA（Si-p53）转染PDLCs，检测低氧下PDLCs中p53表达水平，比较碱性磷酸酶（ALP）活性，成骨标志物ALP、I型胶原（COL1）、成骨特异性转录因子（RUNX2）的mRNA表达量变化。采用SPSS 13.0软件对数据进行统计学分析。

相比正常氧培养后HIF-1α/GAPDH蛋白比值0.309±0.052，PDLCs在低氧培养12、24、48 h后HIF-1α/GAPDH蛋白水平显著升高为0.801±0.049、0.881±0.037与0.936±0.039，差异具有统计学意义（t=6.901、9.041、9.704，P=0.002、0.0008、0.0006）；同时p53/GAPDH蛋白比值分别为0.463±0.036、0.612±0.040与0.858±0.034，相较常氧的0.233±0.035显著上调，差异具有统计学意义（t=4.595、7.140、12.84，P=0.010、0.002、0.0002）。Si-HIF1α转染PDLCs并在低氧培养后，HIF1α-Si1、Si2转染组比阴性NC-Si组的HIF-1α在蛋白水平分别下降64.57%与59.94%，在mRNA水平分别下降66.67%、63.67%，差异具有统计学意义（t蛋白=9.326、6.985，P蛋白=0.0007、0.002；tRNA=5.319、5.015，PRNA=0.006、0.008）；同时p53在蛋白水平分别下降36.47%与38.41%，在mRNA水平分别下降33.43%、30.67%，差异具有统计学意义（t蛋白=4.645、4.135，P蛋白=0.011、0.029；tRNA=4.373、3.912，PRNA=0.012、0.017）。PDLCs经p53-Si1、Si2转染后p53蛋白表达较NC-Si阴性组分别下降56.41%与51.24%，差异具有统计学意义（t=8.194、6.621，P=0.0012、0.0027），但HIF-1α蛋白水平无明显变化，差异无统计学意义（t=1.167、1.391，P=0.308、0.237）。将PDLCs转染p53-siRNA后继续在低氧培养48 h，p53-Si1、Si2组中PDLCs的ALP活性较NC-Si组升高2.05倍与2.17倍，差异具有统计学意义（t=4.889、4.346，P=0.008、0.012）；成骨标志物ALP的mRNA水平分别升高2.14倍与2.05倍，差异具有统计学意义（t=5.423、4.078，P=0.006、0.015）；COL1的mRNA水平分别升高2.86倍与3.03倍，差异具有统计学意义（t=7.56、6.89，P=0.002、0.002）；RUNX2的mRNA水平分别升高3.41倍与3.71倍，差异具有统计学意义（t=8.15、12.21，P=0.001、0.0003）。

低氧可上调HIF-1α与p53表达，进而抑制PDLCs成骨分化。

To investigate the role of p53 in hypoxia induced inhibition of osteogenic differentiation of periodontal ligament cells (PDLCs).

PDLCs were cultured under hypoxia (1% O₂) or normoxia (20% O₂) for 48 h. Western blot was used to detect the expression of p53 and HIF-1α at 12, 24, and 48 h. After transfection with siRNAs targeting HIF-1α (Si-HIF1α), HIF-1α and p53 expression was furthered detected. After transfection with siRNAs targeting p53 (Si-p53), p53 and HIF-1α expression, changes of alkaline phosphatase (ALP) activity, and mRNA expression of osteogenic markers ALP, collagen-I (COL1), and runt related transcription factor 2 (RUNX2) were detected to evaluate osteogenic differentiation of PDLCs under hypoxia. The data were statistically analyzed with SPSS 13.0 software package.

Compared with the value (0.309±0.052) under normoixa, the relative expression of HIF-1α to GAPDH protein in PDLCs under hypoxia for 12, 24 and 48 h was significantly increased to 0.801±0.049, 0.881±0.037, and 0.936±0.039, respectively (t=6.901, 9.041, and 9.704; P=0.002, 0.0008, and 0.0006). The relative expression of p53 to GAPDH protein in PDLCs was significantly increased from 0.233±0.035 under normoixa to 0.463±0.036, 0.612±0.040, and 0.858±0.034 under hypoxia for 12, 24, and 48 h, respectively (t=4.595, 7.140, and 12.84; P=0.010, 0.002, and 0.0002). After PDLCs were transfected with Si-HIF1α and further cultured under hypoxia, HIF-1α expression in the HIF1α-Si1 and HIF1α-Si2 groups was significantly decreased by 64.57% and 59.94% at the protein level, and by 66.67% and 63.67% at the mRNA level compared with the NC-Si group, respectively (t_protein=9.326 and 6.985, P_protein=0.0007 and 0.002; t_RNA=5.319 and 5.015, P_RNA=0.006 and 0.008); p53 expression in the HIF1α-Si1 and HIF1α-Si2 groups was decreased by 36.47% and 38.41% at the protein level, and by 33.43% and 30.67% at the mRNA level, respectively (t_protein=4.645 and 4.135, P_protein=0.011 and 0.029; t_RNA=4.373 and 3.912, P_RNA=0.012 and 0.017). After PDLCs were transfected with Si-p53, p53 expression in the p53-Si1 and p53-Si2 groups was significantly decreased by 56.41% and 51.24% compared with the NC-Si control group (t=8.194 and 6.621, P=0.0012 and 0.0027). However, no significant changes in HIF1α expression were observed in the p53-Si1 and p53-Si2 groups compared with the NC-Si group (t=1.167 and1.391, P=0.308 and 0.237). After PDLCs were transfected with Si-p53 and further cultured under hypoxia for 48 h, ALP activity in the p53-Si1 and Si2 p53-groups was significantly increased by 2.05-fold and 2.17-fold compared with the NC-Si control group (t=4.889 and 4.346, P=0.008 and 0.012); ALP mRNA in the p53-Si1 and p53-Si2 groups was significantly increased by 2.14-fold and 2.05-fold than that of the NC-Si control group (t=5.423 and 4.078, P=0.006 and 0.015); COL1 mRNA was significantly increased by 2.86-fold and 3.03-fold (t=7.56 and 6.89, P=0.002 and 0.002); and RUNX2 mRNA was significantly increased by 3.41-fold and 3.71-fold (t=8.15 and 12.21, P=0.001 and 0.0003).

Hypoxia increases HIF-1α and p53 expression, and hypoxia inhibits osteogenic differentiation of PDLCs via upregulation of p53.