吡咯烷二硫氨基甲酸干预脂多糖刺激下牙周膜干细胞RANKL和OPG的表达

Intervention with pyrrolidine dithioearbamate stimulates expression of RANKL and OPG in lipopolysaccharide-stimulated periodontal ligament stem cells

背景:研究表明,调控破骨细胞、成骨细胞形成与分化的多种细胞因子,如RANKL和OPG主要是由成骨细胞和骨髓基质细胞分泌的,而与骨髓基质细胞同源的牙周膜干细胞在炎症免疫调控过程中也发挥着显著作用,因此极有可能在毒力因子刺激下也参与RANKL和OPG的表达调控,从而介导牙槽骨吸收。目的:探讨采用吡咯烷二硫氨基甲酸(pyrrolidine dithiocarbamate,PDTC)抑制核因子κB活性能否缓解炎症状态下牙周膜干细胞中RSNKL/RANK/OPG系统的失衡状态。方法:将第4代牙周膜干细胞分为4组:(1)对照组:含体积分数为10%胎牛血清的DMEM培养基;(2)脂多糖组:培养基中加入10 mg/L脂多糖;(3)10μmol/L PDTC组:用10μmol/L的PDTC处理细胞30 min,弃去培养基,再加入含10 mg/L脂多糖的培养基;(4)100μmol/L PDTC组:用100μmol/L的PDTC处理细胞30 min,弃去培养基,再加入含10 mg/L脂多糖的培养基。后两组在PDTC处理30 min后倒置显微镜下观察细胞形态,培养24 h后,收集细胞采用RT-PCR检测各组细胞OPG、RANKL mR NA表达量及RANKL/OPG比值变化。结果与结论:(1)PDTC预处理浓度达100μmol/L即可引起细胞形态学改变:细胞贴壁部分回缩,变圆,胞体缩小;(2)与对照组比较,脂多糖组RANKL表达显著升高(P<0.05),10μmol/L PDTC组RANKL表达较脂多糖组无显著性变化(P>0.05),100μmol/L PDTC组RANKL表达较脂多糖组显著降低(P<0.05);(3)与对照组比较,脂多糖组OPG表达略低(P>0.05),10μmol/L PDTC组OPG表达较脂多糖组显著升高(P<0.05),100μmol/L PDTC组OPG表达与脂多糖组比较差异无显著性意义(P>0.05);(4)与对照组比较,其余各组RANKL/OPG比值均有显著升高(P<0.05),脂多糖组升高最明显,随着PDTC浓度升高,RANKL/OPG比值逐渐降低(P<0.05);(5)结果表明,牙周膜干细胞可能在炎症状态下通过改变RANKL及OPG表达量参与调控破骨细胞分化成熟,而PDTC能够通过抑制核因子κB活性缓解炎症状态下牙周膜干细胞中RANK/RANKL/OPG系统的失衡状态。

BACKGROUND： It is generally believed that various cytokines, such as receptor activator of nuclear factor-κB ligand （RANKL） and osteoprotegerin （OPG）, which regulate osteoclast and osteoblast formation and differentiation, are mainly secreted by osteoblasts and bone marrow stromal cells. Whereas in the periodontal tissues, homologous periodontal ligament stem cells （PDLSCs） also play a significant role in the regulation of inflammation and immunity. Thus, virulence factors are very likely to mediate alveolar bone resorption by involvement in the regulation of RANKL and OPG.OBJECTIVE： To explore whether the inhibition of nuclear factor-κB activity by pyrrolidine dithiocarbamate （PDTC） can relieve the imbalance of RANKL/RANK/OPG system in PDLSCs in inflammatory state.METHODS： After isolation, culture and differentiation in vitro, passage 4 PDLSCs were divided into four group： control group, cultured in the DMEM medium containing 10% fetal bovine serum; lipopolysaccharide （LPS） group, cultured in the DMEM medium containing 10 mg/L LPS; 10 μmol/L PDTC group, pretreated with 10 μmol/L PDTC for 30 minutes followed by removal of the medium and addition of the medium containing 10 mg/L LPS; 100 μmol/L PDTC group, pretreated with 100 μmol/L PDTC for 30 minutes followed by removal of the medium and addition of the medium containing 10 mg/L LPS. In the latter two groups, cell morphology was observed under inverted microscope at 30 minutes after PDTC pretreatment. After 24 hours of culture, expression of OPG and RANKL mRNA and the ratio of RANKL/OPG in cells were detected in each group.RESULTS AND CONCLUSION： （1） PDTC pretreatment at a concentration of 100 μmol/L could cause cell morphological changes： some cells were adherent and retracted, and became round with the cell body shrinking in size. （2） Compared with the control group, the expression of RANKL in the PDLSCs was significantly increased in the LPS group （P 〈 0.05）, and the expression of RANKL in the PDLSCs pretreated with 10 μmol/L PDTC had no significant change （P 〉 0.05）, while compared with the LPS group, the expression of RANKL in the PDLSCs pretreated with 100 μmol/L PDTC was significantly lowered （P 〈 0.05）. （3） Compared with the control group, the expression of OPG in the PDLSCs was slightly lowered in the LPS group （P 〉 0.05）. After pretreatment with 10 μmol/L PDTC, the expression of OPG in the PDLSCs was significantly increased （P 〈0.05） compared with the LPS group, while the expression of OPG in the PDLSCs pretreated with 100 μmol/L PDTC had no significant difference from that in the LPS group （P 〉 0.05）. （4） Compared with the control group, the ratios of RANKL/OPG in the other groups were significantly increased （P 〈 0.05）, especially in the LPS group. With the increase of PDTC concentration, the ratio of RANKL/OPG decreased gradually（P 〈 0.05）. These results reveal that PDTC can relieve the imbalance of RANK/RANKL/OPG system in the PDLSC in inflammatory state by inhibiting nuclear factor-κB activity.