脂多糖对体外培养成骨细胞凋亡影响的初步实验研究

Primary experimental study on the influence of LPS on osteoblast apoptosis in vitro

目的探讨脂多糖(Lipopolysaccharides,LPS)对成骨细胞凋亡的影响。方法体外培养成骨细胞作为细胞模型,对照组采用10%的FBS的DMEM/F12培养液培养,干预组在对照组正常培养1 d后加入10mmol/LLPS培养。取第3代成骨细胞按实验设计分组,于LPS作用后第1、3、5和7天进行形态学观察及成骨细胞增殖的检测,于LPS作用后第3、7、10和14天进行成骨细胞分化指标检测,于LPS作用后第1天采用流式细胞仪对成骨细胞凋亡进行检测。结果两组第1天细胞增殖差异无显著性(P>0.05),干预组第3、5和7天细胞增殖率低于对照组,差异有显著性(P<0.05或P<0.01)。培养后第3、7、10和14天,对照组细胞碱性磷酸酶(ALP)、骨钙素(BGP)逐渐升高,干预组细胞ALP、BGP逐渐降低(P<0.05);同期比较,干预组细胞ALP和BGP含量低于对照组(P<0.01)。对照组成骨细胞第1天自发性凋亡率低于干预组(1.61±0.39)%<(3.14±0.60)%,差异有显著性(P<0.01)。结论一定浓度的内毒素可以诱导体外培养成骨细胞发生凋亡,并使其增殖与分化能力逐渐减弱。

[ Objective ] To study the influence of endotoxin on osteoblast apoptosis in vitro. [Methods ] The mouse calvarial osteoblasts were cultured in vitro to three generations and divided into two groups ： the control group and intervention group. Cells of control group were cultured in DMEM/F12 medium containing 10%FBS, to cells of intervention group added 10mmol/L LPS after 1 day normal culture. After 1, 3, 5 and 7 d culture, cells were harvest- ed to measure MTT value. After 3, 7, 10 and 14 d culture, cells were harvested to measure alkaline phosphatase （ALP） and osteocalcin （BGP） activity. After 1 d culture, the osteoblast apoptosis was assessed by flow cytometry. [ Results ] There was no significant difference in the value of MTT between two groups （P 〉0.05）. The cellular prolif- eration rate was significantly lower in intervention group after 3, 5 and 7 d culture （P 〈0.05 or P 〈0.01）. The values of ALP and BGP were significantly increased in control group and decreased in intervention group after 3, 7, 10 and 14 d culture （P 〈0.05）. The values of ALP and BGP were significantly lower in intervention group than those in con- trol group after 3, 7, 10 and 14 d cuhure （P 〈0.01）. The apoptotic rate was significantly higher in intervention group than that in control group after ld culture（P 〈0.01）. [ Conclusion] LPS had a marked effect on osteoblast apoptosis by weakening the proliferation and differentiation.