【作者】 梁莉；

【导师】 丁寅；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2008， 博士

【摘要】 骨密度降低与骨质疏松是骨代谢增龄改变的结果,是中老年骨骼生理的主要特征。绝经后妇女骨质疏松发展迅速,与机体雌激素缺乏有关。研究表明雌激素在骨吸收与改建中发挥了极其重要的作用。牙槽骨是机体骨质密度较低的骨组织,最容易发生骨质疏松,主要表现为牙槽骨吸收,导致牙齿松动与脱落,直接影响患者牙齿和牙周组织健康与口腔治疗,但至今对牙槽骨吸收与改建的机理并不十分明确。人牙周膜成纤维细胞(Human periodontal ligament fibroblast,HPLF)是牙周组织中重要的细胞成分,通过增殖、分化、分泌基质参与到牙周组织的改建中,研究表明雌激素能够促进牙周膜成纤维细胞向成骨分化,增加其成骨活力,对于维持牙周组织正常的改建和损伤后的修复有重要的意义。雌激素的生物学作用主要是通过细胞内雌激素受体(Estrogen receptor,ER)介导的,研究证实牙周膜成纤维细胞有ER基因的表达,但雌激素受体在牙周膜成纤维细胞成骨分化中所起的作用及其机理尚不清楚。因此,为了探讨雌激素受体在牙周膜成纤维细胞成骨分化中的作用,以期进一步探明雌激素受体在牙槽骨吸收与改建中的作用以及骨质疏松牙槽骨吸收的机理,进而为减缓牙槽骨吸收、维护牙齿稳固性开辟新的途径,我们设计并进行了如下实验:1、雌激素受体在人牙周膜成纤维细胞中的表达取因正畸原因拔除的第一前磨牙牙周膜组织,用组织块结合酶消化法培养人牙周膜成纤维细胞,采用RT-PCR法检测ERα和ERβmRNA的表达,Western blot法分析ERα和ERβ蛋白的表达。2、ERβ基因siRNA表达载体的构建及其在人牙周膜成纤维细胞中的表达依据siRNA设计原则,结合ERβ基因的序列特征,设计合成靶向ERβ的siRNA,将其插入pSilencer 3.1-H1-neo载体,构建ERβ基因的siRNA真核表达载体,并对其进行鉴定。应用脂质体法将干涉载体瞬时转染HPLF细胞,应用RT-PCR、Western blot法初步鉴定瞬时转染前后HPLF细胞中ERβ基因及蛋白表达水平有无变化。经G418抗性筛选,获得稳定转染的细胞,应用RT-PCR、Western blot法分别检测稳定转染siRNA真核表达载体前后HPLF细胞中ERβmRNA和蛋白水平的变化。3、雌激素受体β对人牙周膜成纤维细胞成骨分化能力的影响以稳定转染的HPLF(HPLF-siERβ1和HPLF-siERβ2)和未转染的HPLF为研究对象,在雌激素刺激下,检测细胞碱性磷酸酶(Alkaline phosphatase,ALP)活性和骨钙素(Osteocalcin,OCN)含量,研究雌激素及雌激素受体对HPLF细胞成骨分化能力的影响。4、雌激素受体β对人牙周膜成纤维细胞OPG和RANKL表达的影响以稳定转染的HPLF(HPLF-siERβ1和HPLF-siERβ2)和未转染的HPLF为研究对象,雌激素干预后,RT-PCR、Western blot分别检测OPG、RANKL mRNA和蛋白的表达,研究雌激素对HPLF细胞OPG和RANKL表达的影响,以及ERβ基因干涉后,细胞OPG和RANKL表达的变化情况。结果发现:1、ERα、ERβmRNA和蛋白在HPLF细胞中均有表达,而且ERβ的表达明显高于ERα。2、通过测序验证,成功构建了ERβ基因siRNA真核表达载体。RT-PCR和Western blot法检测分别表明瞬时转染的HPLF中ERβ基因的表达、蛋白含量均受到明显抑制。经过G418筛选获得稳定转染的HPLF细胞,RT-PCR、Western blot检测结果再次表明,siRNA明显抑制了HPLF细胞中ERβ基因的表达。3、雌激素对细胞干预后,未转染的HPLF细胞ALP活性和OCN含量明显增强,而稳定转染的HPLF细胞ALP活性和OCN含量没有明显改变。4、雌激素干预细胞后,未转染的HPLF细胞中OPG mRNA和蛋白的表达显著增强,RANKL mRNA和蛋白的表达显著减少,但稳定转染的HPLF细胞中OPG和RANKL mRNA和蛋白的表达没有明显变化。结论:1、体外培养的人牙周膜成纤维细胞中可观察到ER两种亚型的表达。RT-PCR和Western blot法分别从mRNA水平和蛋白水平证实了ERα、ERβ在HPLF上均有表达,且ERβ的表达明显强于ERα,提示雌激素在HPLF细胞的增殖、分化中发挥作用是通过配体-受体途径实现的,而且可能主要是以ERβ为主介导的。2、ERβsiRNA表达载体能够有效抑制HPLF细胞中ERβmRNA和蛋白水平的表达。3、雌激素明显增加了未转染的HPLF细胞ALP活性和OCN的含量,但对ERβ基因干涉的HPLF细胞ALP和OCN的表达没有明显影响。说明雌激素可能主要是通过ERβ亚基促进牙周膜成纤维细胞向成骨方向分化,增强了牙周膜成纤维细胞的成骨活力。4、HPLF细胞在雌激素作用下,其OPG的表达显著提高,RANKL表达明显减少,但HPLF细胞经ERβsiRNA转染封闭ERβ亚基后,雌激素对OPG和RANKL表达水平没有明显的影响。提示雌激素可能主要通过ERβ下调牙周膜成纤维细胞的RANKL/OPG浓度比,影响OPG/RANKL/RANK信号系统,从而抑制破骨细胞分化,刺激骨形成和抑制骨吸收,在防止牙槽骨吸收中起到重要的作用。更多还原

【Abstract】 Insufficient estrogen is believed to be one of the major causes of postmenopausal osteoporosis. Osteoporosis is considered as one of the risk factors for periodontal disease and tooth loss. A number of studies have suggested that estrogen may have an important role in chronic inflammatory periodontal diseases. It has been observed that gingival inflammation and hyperplasia frequently occur during puberty, pregnancy, and menstruation; gingival inflammation is also frequently observed in women taking oral contraceptives. However, the relationship between the levels of estrogen and the incidence and progression of periodontal diseases is yet to be clarified, partly because the precise effects of estrogen on periodontal tissues are not yet known.The periodontal ligament (PDL) is the connective tissue located between the alveolar bone and the root surface of the tooth. Cells of this ligament exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. They play an important role in maintaining the integrity of the periodontal tissue.Estrogen has been reported to increase alkaline phosphatase activity, the production of osteocalcin, and the formation of mineralized nodules in cultured PDL cells. Estrogen modulates the activity of target cells by binding specific intracellular estrogen receptors (ER). Two estrogen receptor subtypes, designated ERαand ERβ, have been identified. As estrogen plays an important role in maintaining normal bone turnover, the genes for ERαand ERβhave been considered as potential candidate genes that might influence bone mass and osteoporotic risk. In previous studies, PDL cells were shown to express the mRNA for ER. However, the effect of ER in estrogen-induced osteoblastic differentiation function of PDL cells are still poorly understood.In terms of this context we designed and carried out the experiment below in order to investigated the effect of ERβin estrogen-induced osteoblastic differentiation function of PDL cells, to help to find the signal transduction pathways that mediate cellular responses to estrogen and any possibilities of new countermeasures to avoid the alveolar bone resorption.1、The expression of ERs in hPDL cellsBy the culture of human periodontal ligament cells, we investigated ERs expression by RT-PCR and supported by western blot analysis.2、Construction of ERβsiRNA expression vector and its expression in hPDL cellsHairpin siRNA templates were designed based on ERβgene sequence and mRNA structure and were cloned into eukaryotic expression plasmid, then confirmation them by DNA sequencing. Using lipidosome method, hPDL cells were transfected with the siRNA recombinant vector. Stable clones cells were obtained after G418 screening. Inhibition effect of ERβmRNA and protein expression were detected by RT-PCR and western blot respectively. 3、Effect of ERβon the osteoblastic differentiation function of hPDL cellsStable transfected and nontransfected cells were cultured with a saturating concentration of 17β-estradiol (10-7 mol/L). ALP activity was analysed and the amount of OCN was assessed.4、Effect of ERβon OPG and RANKL expression in hPDL cellsStable transfected and nontransfected cells treated with or without 17β-estradiol (10-7 mol/L) for 48h. The protein and corresponding mRNA level of OPG and RANKL expression in the cells were quantitatively determined by western blot and RT-PCR.Results:1、After successful culture of hPDL cells, both ERαand ERβexpression were found in the cells by RT-PCR and western blot and the expression of ERβwere stronger than ERα.2、Eukaryotic expression plasmid expressing siRNA targeting ERβgene was constructed successfully. The results of the RT-PCR and western blot analysis showed that ERβmRNA and protein expression were inhibited and stabilized transfected cells were constructed successfully.3、Estradiol significantly enhanced the ALP activity and the production of OCN in hPDL cells. However, the ALP activity and the production of OCN in hPDL-siERβcells were not significantly changed after estradiol treatment.4、Estradiol caused an increase in OPG expression and decreased RANKL expression in hPDL cells. However, estradiol had no effect on the expression of OPG and RANKL in hPDL-siERβcells.Conclusion:1、Both ERαand ERβexpression were expressed in hPDL cells, providing the bases that the ERs may be involved in the pathology mechanism of alveolar bone loss in postmenopausal women.2、The ERβsiRNA plasmid was constructed successfully and ERβexpression of hPDL cells was inhibited by RNAi.3、ERβmay play important roles in estrogen-induced effects on osteoblastic differentiation function of hPDL cells and estrogen influences the bone formation capacity of hPDL cells mainly via ERβ.4、Estrogen may play an important role in exerting antiresorptive effects on alveolar bone, at least in part, by increasing the expression level of OPG versus that of RANKL via ERβin hPDL cells.更多还原