CPT诱导外周血破骨细胞凋亡作用及其分子机制研究

CPT induced apoptosis of peripheral blood derived osteoclasts and its molecular mechanism

目的:探讨环化变构肿瘤坏死因子相关凋亡诱导配体(CPT)诱导外周血破骨细胞凋亡作用及其分子机制。方法:外周血单个核细胞(PBMC)经巨噬细胞集落刺激因子(M-CSF)及可溶性核因子κB受体活化因子配体(sRANKL)诱导培养成熟破骨细胞,采用不同浓度的CPT诱导破骨细胞凋亡,应用Annexin V-FITC荧光染色法检测CPT诱导破骨细胞凋亡,RT-PCR检测破骨细胞中CPT受体mRNA表达变化的影响。结果:PBMC在RANKL和M-CSF细胞因子诱导下培养21 d,均生成了具有骨吸收活性的破骨细胞。在2×10^6的接种密度下诱导产生破骨细胞的数量(细胞因子含量:30 ng/mL RANKL和30 ng/mL M-CSF)在5×10^5接种密度下(细胞因子含量:30 ng/mL RANKL和30 ng/mL M-CSF)的2倍。通过抗酒石酸酸性磷酸酶(TRAP)染色后,不同浓度(0、100、500 ng/ml)CPT作用正常人的破骨细胞24 h和48 h后,出现明显的细胞凋亡特征,Annexin V-FITC染色后,经荧光显微镜观察,0 ng/ml CPT诱导的对照组极少细胞被染色(绿色荧光);对比发现100、500 ng/ml CPT诱导的给药组中出现大量绿色荧光阳性细胞,进一步研究显示,100、500 ng/ml CPT处理破骨细胞24 h后,破骨细胞凋亡率由1.7%(0 ng/ml)上升到9.1%(100 ng/ml)和13.7%(500 ng/ml),差异有统计学意义(P<0.05);处理48 h后,由2.1%(0 ng/ml)上升为13.8%(100 ng/ml)和21.9%(500 ng/ml),差异有统计学意义(P<0.05),并且发现48 h凋亡数量明显多于24 h(P<0.05)。不同浓度的CPT(100 ng/ml、500 ng/ml)处理破骨细胞24 h后,TRAIL的相关死亡受体4(DR4)、死亡受体5(DR5)、诱骗受体1(DcR1)及诱骗受体2(DcR2)的mRNA表达差异无统计学意义(P>0.05),处理48 h后,DR5的mRNA表达量由1.20±0.26(0 ng/ml)下降为1.00±0.26(100 ng/ml)、0.60±0.09(500 ng/ml),差异有统计学意义(P<0.05)。结论:CPT能以时间-浓度依赖性的诱导外周血源破骨细胞凋亡,其作用机制可能是通过下调DR5的mRNA表达水平诱导其凋亡。

Objective:To investigate the effect of CPT on peripheral blood osteoclast apoptosis and its molecular mechanism. Method:The mature osteoclasts were induced by M-CSF and RANKL in peripheral blood mononuclear cells.Different concentrations of CPT were added to induce the apoptosis of osteoclasts.Annexin V-FITC fluorescence staining was used to detect the apoptosis of osteoclasts induced by CPT.RT-PCR was used to detect the expression of CPT receptor mRNA in osteoclasts. Result:PBMC were induced by RANKL and M-CSF cytokines for 21 days,and osteoclasts with bone resorption activity were produced.The number of osteoclasts(cytokine content:30 ng/mL RANKL and 30 ng/mL M-CSF)induced at 2×10^6 inoculation density was about twice that at 5×10^5 inoculation density(cytokine content:30 ng/mL RANKL and 30 ng/mL M-CSF).After TRAP staining,osteoclasts treated with different concentrations of CPT(0,100,500 ng/ml)for 24 hours and 48 hours showed obvious apoptosis characteristics.Annexin V-FITC staining fluorescence microscopy showed that only a small number of cells in the control group(0 ng/ml)showed green fluorescence,while those in the experimental group(100,500 ng/ml)showed a large number of green fluorescence positive cells.Further studies showed that the apoptosis rate of osteoclasts increased from 1.7%(0 ng/ml)to 9.1%(100 ng/ml)and 13.7%(500 ng/ml)after 24 hours of treatment with 100 and 500 ng/ml CPT,and the difference was statistically significant(P<0.05).The number of apoptotic cells increased to 13.8%(100 ng/ml)and 21.9%(500 ng/ml)in 48 hours(P<0.05).There was no significant difference in the mRNA expression of DR4,DR5,DcR1 and DcR2 in osteoclasts treated with different concentrations of CPT(100 ng/ml,500 ng/ml)for 24 hours(P>0.05).After 48 hours,the mRNA expression of DR5 decreased from 1.20±0.26(0 ng/ml)to 1.00±0.26(100 ng/ml)and 0.60±0.09(500 ng/ml)with significant difference(P<0.05).Conclusion:CPT can induce the apoptosis of osteoclasts from peripheral blood in a time-concentration dependent manner,and the mechanism may be through down-regulating the mRNA expression of DR5.