雌激素受体β对牙周膜细胞骨向分化能力影响的研究

目的 研究小干扰RNA(small interfering RNA,siRNA)抑制人牙周膜成纤维细胞(human periodontal ligament fibroblast cell,HPLF)中雌激素受体β(estrogen receptor β,ERβ)后,对17β-雌二醇(E2)诱导的成骨能力的影响。方法 设计并合成针对ERβ基因siRNA的寡核苷酸序列,插入载体形成重组载体。重组载体经测序鉴定后,以脂质体法转染至HPLF细胞中,蛋白质印迹法及RT-PCR法检测转染前后ERβ的表达情况。鉴定后用1×10-7mol/L的17β-E2干预经ERβsiRNA转染的HPLF细胞和未经ERβsiRNA转染的HPLF细胞,测定细胞的碱性磷酸酶(alkaline phosphatase,ALP)活性和骨钙素(os-teocalcin,OCN)含量。结果 测序鉴定重组质粒后,蛋白质印迹法及RT-PCR结果 显示ERβsiRNA载体可特异地抑制HPLF细胞中ERβ的表达。经雌激素干预后,HPLF细胞ALP活性和OCN含量高于对照组(P<0.01),但ERβsiRNA载体稳定转染的HPLF细胞ALP活性和OCN含量与对照组相比没有显著性差异。结论 雌激素可能通过ERβ亚基促进人牙周膜成纤维细胞的骨向分化能力。

Effects of estrogen receptor beta on osteoblastic differentiation function of human periodontal ligament cells

Objective To investigate the effects of ERβ on osteoblastic differentiation function of HPLF cells by measuring the alkaline phosphatase（ALP） activity and the production of osteocalcin（OCN） in vitro.Methods We employed a short interfering RNA（siRNA） technique to inhibit ERβ expression in HPLF cells.The cells were cultured with a saturating concentration of 17β-estradiol（10-7 mol/L）.ALP activity was analysed by colorimetric assay using ALP kit and the amount of OCN was assessed by osteocalcin ELISA kit.Results The results of the RT-PCR and Western blot analysis showed that ERβ mRNA and protein expression were inhibited and stabilized transfected cells were constructed successfully.A clear increase of ALP activity and the levels of OCN were observed after estradiol incubation in HPLF cells（P0.05）.However,no evident changes of ALP activity and the levels of OCN could be observed in HPLF-siERβ cells after estradiol treatment.Conclusions These results indicate that estrogen may influence the bone formation capacity of HPLF cells mainly via ERβ.