【作者】 曹猛；

【导师】 丁寅；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2007， 博士

【摘要】 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,雌激素缺乏可以引起全身骨质疏松。牙槽骨是机体骨质密度较低的骨组织,最容易出现骨质疏松,临床上主要表现为牙槽骨吸收,导致牙齿松动与脱落,直接影响患者牙齿与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。牙槽骨骨质疏松及吸收是否受雌激素缺乏的影响,目前的报道尚有争论,使得研究骨质疏松状况下牙周组织的生理、病理特点成为涉及牙周病学、牙槽外科、牙齿种植、口腔修复及正畸治疗等口腔多学科的临床问题,也是本研究所关注的问题。人牙周膜中的成纤维细胞( Human Periodontal Ligament Fibroblast,HPLF)是最主要的细胞成分,牙周膜成纤维细胞通过增殖、分化、分泌基质参与到牙周组织的改建中,在维持牙周组织完整性以及行使牙周改建的功能中发挥重要的作用,而雌激素在其中发挥的作用是我们关心的问题。国内外学者的研究提供了雌激素对HPLF增殖、分化影响的一些线索,如雌激素作用下牙周膜成纤维细胞可以表现出一定的成骨特性,例如分泌骨相关蛋白,以及形成矿化结节等,但其作用途径尚未阐明。雌激素受体(estrogen receptor,ER)是雌激素的细胞内结合位点,是雌激素发挥作用的关键所在。在机体其他组织和器官,雌激素通常是通过与其特异性受体结合发挥调控细胞生长、增殖和分化的。ER的两种亚型ERα和ERβ已经成功分离出来,在不同的组织中有不同的表达型。目前国内外关于ER与牙周组织关系方面的研究较少。雌激素可能能够影响牙周膜成纤维细胞的一些生物学特性,例如蛋白合成和细胞生长,也可能对成纤维细胞成骨分化有一定影响,通过本研究我们希望进一步探讨雌激素对牙周改建的生理作用,为临床正畸治疗和牙周病的治疗提供一定的实验依据。我们通过(1)切除SD大鼠两侧卵巢(OVX),建立人工模拟绝经后的动物模型,观察大鼠牙槽骨高度(临床附着丧失)和血清雌激素水平的关系;(2)取大鼠下颌磨牙区组织,制备冰冻切片后,用免疫组织化学SABC法进行牙周组织ERα、ERβ染色,计数随机区域中阳性细胞个数并进行统计学分析。(3)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牙周膜成纤维细胞,制作细胞爬片,免疫细胞化学进行ERα、ERβ染色,逆转录聚合酶链式反应(RT-PCR)法检测ER的mRNA的表达,Western blot法分析ER蛋白的表达;(4)对体外培养的牙周膜成纤维细胞给与不同生理浓度的雌激素刺激,以3H-胸腺嘧啶和3H-脯氨酸参入实验确定细胞DNA和胶原合成产物的变化,明确雌激素对细胞增殖的影响;(5)在不同生理浓度的雌激素刺激下,测定细胞碱性磷酸酶活性(ALP)和骨钙素(OCN)含量,明确雌激素对细胞骨分化能力的影响。研究结果发现:(1)去势组大鼠下颌第二磨牙区可见牙槽骨皮质变薄,骨小梁变稀疏,排列紊乱,骨髓腔隙增大,骨小梁表面成骨细胞及破骨细胞均有增加。牙周膜水肿、疏松,排列不规则,血管扩张充血,牙骨质密度也有减低,较假手术组明显呈退行性变,临床附着水平也与假手术组有统计学差异(P<0.01),而雌激素治疗组可改善这一病理变化。这与动物血清雌激素浓度有明显的相关性。(2)大鼠牙周膜组织切片中,可见ER阳性染色的棕黄色颗粒,PBS空白对照组则无棕黄色染色,阳性对照可见强阳性染色。观察发现ERα、ERβ在SD大鼠牙周组织中比较广泛的表达,ER在细胞胞核和胞浆均有表达,ERβ的阳性率明显高于ERα。(3)体外培养人牙周膜成纤维细胞制备细胞爬片,通过免疫细胞化学方法可观测到ERα、ERβ在胞核中表达。(4)通过RT-PCR检测ERmRNA,各组细胞用雌激素受体的特异性引物均获得了目的PCR产物,分别对应ERα、ERβ的特异条带(5)同株细胞经Western blot检测结果,各组细胞可见正确分子量的蛋白印迹条带,其中ERβ的条带较ERα对应的蛋白条带浓。(6)以10-7, 10-8和10-9mol/L浓度的E2作用于细胞,与阴性对照和空白对照组相比,各种浓度的雌激素均未增加细胞DNA的合成量和胶原合成(P>0.05)(7)体外培养牙周膜成纤维细胞用浓度为10-7、10-8、10-9mol/L的E2分别处理,与空白对照组相比,E2浓度依赖地增加细胞内ALP活性和OCN的含量,受体特异性阻断剂ICI182780可以阻断这一效应。根据以上研究结果,得出以下结论:(1)雌激素缺乏时动物牙槽嵴高度降低,骨量减少,附着丧失增加,而给予去势后大鼠补充雌激素,牙槽骨的骨密度增加,附着丧失减轻,表明雌激素对于改善骨质疏松具有一定作用。(2)大鼠牙周膜中,可观察到ER免疫组化阳性的细胞,结合细胞分布特点,可以得出成纤维细胞中有雌激素受体表达的结论。说明牙周组织对雌激素有直接应答反应。ERα和ERβ阳性率分别为20%和40%左右,二者间有显著性差异(P<0.05),说明在大鼠的牙周膜以ERβ的表达为主。(3)体外培养的人牙周膜成纤维细胞中也可观察到ER两种亚型的表达。RT-PCR法和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。提示雌激素在HPLF的增殖、分化中发挥作用很可能是通过配体—受体途径实现的(4)生理浓度的雌激素对牙周膜成纤维细胞的胶原和DNA合成没有影响,说明雌激素-受体复合体作用的基因可能与细胞的增殖和胶原合成无关。(5)生理浓度的雌激素能促进牙周膜成纤维细胞合成和释放细胞成骨分化的指标ALP和OCN,说明雌激素能够促进牙周膜成纤维细胞向成骨方向分化,这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。更多还原

【Abstract】 Estrogen deficiency, which is important in the pathogenesis of postmenopausal osteoporosis, has received increasing attention in the studies related to the periodontal diseases in postmenopausal women, such as the alveolar bone resorption and clinical attachment loss. Accumulating evidences have demonstrated that osteoporotic women exhibited a higher frequency of bone turnover, as observed in the femur. In the ovariectomized rat model, estrogen induced similar activation of bone resorption with increased. Although there is no doubt that estrogen has a broad influence in the biology of various human tissues, the etiology of estrogen-associated periodontal diseases remains an enigma. Therefore, we hypothesize that estrogen may play an important role in the maintenance and regeneration of periodontal tissue.The periodontal ligament is the connective tissue located between the alveolar bone and root surface of the tooth. Periodontal ligament cells (PDLcs) play an important part in maintaining the integrity of the periodontal tissue. PDLcs are capable of differentiating into osteogenic cells and exhibit osteoblastic phenotypes, such as the production of bone-like matrix proteins, high alkaline phosphatase (ALP) activity, and the formation of calcified nodules. It is well known that estrogen affects the biological properties of osteogenic cells such as the production of osteocalcin and ALP activity. However, less information is available regarding the effect of estrogen on PDLcs. Better understanding the mechanisms of estrogen on periodontium would facilitate our effort in the maintenance of the health of postmenopausal periodontal tissue.The sex hormones exert their influence on target tissues by binding to specific receptors that belong to a superfamily of ligand-activated transcription factors that regulate cell growth, differentiation and development. Two estrogen receptor (ER) subtypes, designated ER-alpha and ER-beta, have been identified and these receptors show a tissue specific expression pattern. When investigating the effects of estrogen on PDLc, it is critical to exam whether ER is expressed in PDLc. Since human osteoblasts express ERs, it is possible that PDLcs also do so. Since it was quite controversial that whether PDLcs express ERs in previous reports, more evidences are needed to assess the expression and functional significance of ER in PDLcs.In the present study, we established animal model by bilateral ovariectomy (OVX) and examined the effect of estrogen deficiency on the alveolar bone metabolism and the change of serum estrogen; investingated the ERs expression in periodontal ligament of rat by histoimmunochemistry. By the culture of human periodontal ligament cells ,we investigated ERs expression in human PDLc by cytoimmunochemistry, ERs mRNA expression by RT-PCR and ERs protein expression by western blotting, and further evaluated the influence of estrogen on DNA and collagen synthesis and the potential action of estrogen on bone-forming in PDLc.We found that:(1) Significant alveolar bone loss was detected in OVX group, indicating the success of the animal model. Estrogen replacement treatment (ERT) clearly attenuated the bone loss by the deficiency of estrogen induced by OVX. In rats of OVX + vehicle, as expected, the average serum 17-beta estradiol level was significant lower than that in OVX + 17-beta estradiol and sham.(2) By histoimmunochemistry, ERαand ERβwere abundantly expressed in periodontal ligament, and ERβwas much more than ERα(p<0.01). ERs were localized in both nuclei and cytoplasms, which indicates estrogen may play its role by different mechanisms in periodontium.(3)After successful culture of primary human periodontal ligament cells, both ERαand ERβprotein expressions were found in the cells by specific antibody by cytoimmunochemistry.(4)ERs mRNA was examined by RT-PCR. The expected PCR products of ER-alpha in 287bp and ER-beta in 659bp respectively were obtained from the primary cultured PDLcs. We found that the PDLcs did express both ER subunit ER-alpha and -beta although the intensities of the HER-alpha and -beta bands were much weaker than those observed in positive control MCF-7 cells. The results suggested that ER mRNA expression in PDLcs is in relative low level.(5)The protein expressions of ERs were also determined by Western Blot analysis. Similar expressions in low levels of ERs were detected compared to the positive control of MCF-7 cell extract. However, no difference was detected in ERs expression after 17-beta estradiol treatment.(6)When PDLcs were incubated in the presence of 17-beta estradiol, neither periodontal ligament cell DNA synthesis nor collagen synthesis was affected by E2 treatment for 24 hours.(7) Both ALP activity and osteocalcin production were increased by 17-beta estrodiol in a dose-dependent manner. Estrogen receptor antagonist ICI 182780 (10-6mol/L) completely blocks the effect of 17-beta estradiol. The results suggested that PDLcs mediates estrogen bone-sparing action through ER, therefore contributes to the mechanisms of estrogen deficiency alveolar bone loss.In conclusion:(1)Our results suggested that estrogen deficiency induced by ovariectomy significantly accelerated alveolar bone loss whereas ERT could efficiently reverse the process,confirmed that there existed obvious correlation between estrogen level and periodontal health.(2) both ER-alpha and ER-beta were expressed in PDLcs providing the bases that the ERs may be involved in the pathology mechanism of alveolar bone loss in postmenopausal women.(3) There was no effect on periodontal ligament cell DNA synthesis and collagen synthesis by treatment with E2. The genes regulated by the estrogen–ER complex in periodontal ligament cells remain to be identified these genes are probably not associated with periodontal ligamentcell proliferation and collagen formation.(4) estrogen could exhibit obvious positive modulation on ALP activity and osteocalcin production in PDLcs,confirmed that estrogen could significantly promote the osteogenic capability of human PDLcs(5)Our current study suggested that estrogen played an important role in periodontal diseases. Further studies will be necessary to delineate the actions of estrogen on periodontal ligament cells in detail.更多还原