【作者】 潘峰；

【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】 绝经后妇女由于雌激素水平下降,易于引发骨质疏松,对骨质疏松与颌骨、牙槽骨关系的研究表明,骨质疏松症的绝经后妇女下颌骨量与全身骨量有明显的相关关系,绝经后可出现不同程度的牙槽骨骨质疏松症状[1-4]。雌激素替代疗法(estrogen replacement treatment,ERT)可有效提高绝经期妇女体内雌激素的水平,是预防绝经后骨质疏松症及骨折的有效方法[2],同时,ERT也可减少牙周炎患者的牙龈出血和附着丧失,维持牙槽骨的骨量,提高牙齿的保留率[5,6]。牙周膜细胞是牙周膜内的主要成分,对维持牙周膜的形态、功能有决定性的作用,并且在牙槽骨的改建中发挥重要作用。以往的研究显示牙周膜细胞内有雌激素受体的表达,而且在外源性雌激素作用下,牙周膜细胞的成骨作用增强[7]。在牙周膜内有数种细胞成分,除占主要地位的牙周膜成纤维细胞外,还有成骨细胞、破骨细胞及未分化间充质细胞--牙周膜干细胞(PDLSCs),PDLSCs是牙周膜内各主要功能细胞的来源[8],而PDLSCs与雌激素在牙周改建及再生过程中的关系尚无研究报道。由此,我们设想,牙周膜干细胞内是否也有雌激素受体的表达,如果有雌激素受体表达,外源性雌激素对牙周膜干细胞成骨作用有何影响?通过本研究,我们将对这些问题进行探讨,以进一步明确雌激素在牙周改建及再生中的作用机制,为牙周病及正畸临床治疗提供一定新的思路和理论基础。实验内容:(1)收集临床拔除的正常人牙周健康、无龋的新鲜第三磨牙,刮取根中1/3牙周膜组织进行牙周膜原代细胞培养。取第四代人牙周膜细胞,通过免疫磁珠技术,以复合间充质干细胞的表面标记物STRO-1抗体的磁珠对人牙周膜干细胞进行筛选、扩增培养,MTT法测定PDLSCs生长曲线并与牙周膜细胞进行比较。(2)取第三代PDLSCs进行克隆形成率、细胞周期分析,对其生物学行为进行研究;以流式细胞仪对PDLSCs及牙周膜细胞进行细胞表面特异性抗原CD34、CD44、CD45、CD146表达测定;以免疫细胞化学方法对PDLSCs进行干细胞表面特异性抗原STRO-1及间充质细胞相对特异性抗原Vimentin表达测定;PDLSCs成脂及成骨多向诱导分化研究。通过上述实验对所分离的PDLSCs干细胞特征进行鉴定。(3)取第三代PDLSCs以免疫细胞化学方法进行ERα及ERβ特异性抗体染色;以逆转录聚合酶链式反应(RT-PCR)法检测PDLSCs在雌激素作用前后ERα及ERβmRNA表达的变化并与牙周膜细胞作比较研究;以Western blot法分析PDLSCs在雌激素作用前后ERα及ERβ蛋白表达的变化并与牙周膜细胞作比较研究。(4)取第三代PDLSCs进行成骨诱导,加入不同浓度梯度(10-7、10-8、10-9M)的外源性雌激素(17β-雌二醇),MTT法测定成骨分化过程中的PDLSCs在雌激素作用下的细胞增殖变化情况。(5)取第三代PDLSCs进行成骨诱导,加入不同浓度梯度(10-7、10-8、10-9M)的外源性雌激素(17β-雌二醇),并以ER抑制剂ICI182780作对照,进行碱性磷酸酶(ALP)表达变化及羟脯氨酸含量的检测。结果:(1)所筛选PDLSCs呈类梭形外观,形态不规则,核为卵圆形、位于胞质中央,似成纤维样细胞。细胞生长曲线示可见PDLSCs较牙周膜细胞生长慢,具有慢增殖周期的干细胞特点。(2)PDLSCs生物学研究显示其具备克隆形成能力及干细胞细胞周期特点,流式分析显示所分离PDLSCs高表达间充质干细胞表面标记物CD44及CD146,内皮细胞及造血系细胞表面标记物CD34和CD45表达极低;干细胞表面特异性抗原STRO-1及间充质细胞相对特异性抗原Vimentin染色均为阳性,此外,PDLSCs经成脂及成骨诱导获得了茜素红染色阳性的类成骨细胞和油红-O染色阳性的类脂肪细胞。(3)免疫细胞化学染色显示ERα及ERβ在PDLSCs中均阳性表达,定位于细胞核;RT-PCR及Western blot检测示:在mRNA及蛋白水平,均可检测到ERα及ERβ表达,且ERα表达高于ERβ。此外,PDLSCs与同来源牙周膜细胞相比, ERα及ERβ的表达较高;而PDLSCs在雌激素作用后ERα及ERβ的表达较作用前均增加。(4)在17-β雌二醇作用下,PDLSCs的增殖发生变化,与对照组相比,前3天雌激素干扰组细胞增殖受到抑制,此后雌激素干扰组细胞增殖高于对照组,在不同药物浓度组间,1×10-7M组对细胞增殖影响最为明显,其细胞增殖水平高于其余两组。(5)在17-β雌二醇作用下,PDLSCs碱性磷酸酶(ALP)的表达发生动态变化,自第三天开始,各组ALP表达均升高,而雌激素干扰组ALP表达量高于对照组,在不同药物浓度组间1×10-7M组ALP表达增加高于其余两组。(6)在17-β雌二醇作用下,PDLSCsⅠ型胶原合成表达与对照组相比有增加趋势,且与药物浓度有剂量依赖关系,在不同药物浓度组间1×10-7M组作用最为明显。结论:(1)用复合间充质干细胞特异性抗体STRO-1的免疫磁珠可自牙周膜细胞中成功筛选PDLSCs,所筛选PDLSCs具有克隆形成、慢细胞周期、表达干细胞特异性标记物及多向分化能力等干细胞特点。(2)PDLSCs中ERα及ERβ均有表达,ERα表达高于ERβ,提示雌激素对PDLSCs的调控作用可能主要通过ERα途径;在外源性雌激素作用下,成骨分化过程中ERα及ERβ表达均增加,提示PDLSCs可能是雌激素调控牙周改建的上游细胞,雌激素通过其受体对PDLSCs的成骨分化进行调控。(3)雌激素可促进PDLSCs的增殖,并对PDLSCs成骨分化过程有促进作用,该作用与ER及雌激素浓度密切相关,提示雌激素可以通过PDLSCs促进牙周改建及再生,对牙周损伤的恢复有积极的作用。更多还原

【Abstract】 Estrogen deficiency is important in the pathogenesis of postmenopausal osteoporosis. Previous studies show that the bone mass in the mandible and skeletal bone mass had an evident correlation in postmenopausal women with osteoporosis, and the osteoporotic alveolar bone would appear in postmenopausal women. Estrogen replacement treatment (ERT) can increase the estrogen level in postmenopausal women and decrease the bone loss, gingival blooding and attachment loss. Estrogen exerts its influence by binding to specific receptors, estrogen receptor (ER). ER-αand ER-βhave been detected in periodontal ligament (PDL) cells, and it has been reported that estrogen could stimulate bone formation capacity in cultured PDL cells by increasing alkaline phosphatase activity, the production of osteocalcin and the formation of mineralized nodules.Periodontal ligament stem cells (PDLSCs) are progenitors of PDL cells, and the application of PDLSCs may be effective for periodontal regenerative therapy as for their ability to form different tissue types and self-renew. However, considering the effects of estrogen on bone-forming capability in cultured human PDL cells, few studies have been conducted in PDLSCs. The aims of the present study were to determine ERs expression in human periodontal ligament stem cells and to investigate the effects that estrogen on osteogenic differentiation of PDLSCs in vitro.In the present study, PDLSCs were isolated by immunomagnetic method. The flow cytometry and immunocytochemistry procedure were used to disclose the cell cycle and surface marker of the PDLSCs. And osteoinduction and adipoinduction were done to conform the multidirectional differentiation ability of PDLSCs as meanwhille. We investigated ERs expression in PDLSCs by immunocytochemistry, ERs mRNA expression by RT-PCR and ERs protein expression by Western Blot analysis. Furthermore, we evaluated the effects of estrogen on ALP activity and collagen synthesis in the osteogenic differentiation of PDLSCs.We harvested the following results:1. The isolated PDLSCs have orbicular-ovate nuclei and irregular morphous, which are similar to fibroblast-like cells. The cell growth curve indicated that PDLSCs had a low proliferation rate.2. The isolated PDLSCs have the ability to form multi-cell clusters and cell life cycle which similar to that of stem cells. Analysis of surface epitopes of PDLSCs by FACS showed CD44 and CD146 were of high expression, in the contrary, CD34 and CD45 were of low expression. In the meanwhile, STRO-1 and Vimentin were both positively stained. Furthermore, after adipogenetic induction, PDLSCs developed into oil red O-positive lipid-laden fat cells as well as small round alizarin red-positive nodules were also formed in the osteogenic induction cultures.3. Immunocytochemistry showed that both ER-αand ER-βwere positively stained in the nuclei of PDLSCs. Western blot analysis and RT-PCR procedure indicated that the expressions of ERs (both ER-αand ER-β) in PDL cells were in a lower level compared with that in the PDLSCs. In PDLSCs groups, the expression of ER-αwas higher than that of ER-β, and the expressions of ERs in 17-βestradiol treated group were higher than that of untreated group.4. Comparing to the group of control, the DNA synthesis of PDLSCs depressed in the first three days, and increased apparently in the following time while treated with 17-βestradiol. In the estrogen treated groups, the DNA synthesis was of a dose dependent manner.5. When PDLSCs were treated with 17-βestradiol, the ALP expression increased from day 3. Furthermore, the ALP levels were higher in the estrogen treated groups than control group, and in the estrogen treated groups, the ALP activity was of a dose dependent manner.6. When PDLSCs were treated with 17-βestradiol, compared with control groups, the synthesis of typeⅠcollagen increased more apparently in the estrogen treated groups, and the synthesis was of a dose dependent manner.Conclusion:1. Immunomagnetic method is an effective way to isolate and purify the PDLSCs. The acquired cells showed the characteristics of stem cells.2. Both ER-αand ER-βare expressed in PDLSCs. The expression of ER-αis higher than ER-β, which indicates that estrogen effects on PDLSCs are probably mainly displayed via ER-α. When PDLSCs were treated with estrogen while osteogenic induction, the expression of ER-αand ER-βincreased, this signified that estrogen may modulate the osteogenic differentiation of PDLSCs through ERs.3. Estrogen could accelerate the DNA synthesis and osteogenic differentiation of PDLSCs, which indicates that the estrogen effects on periodontal reconstruction may be displayed through PDLSCs.更多还原