【作者】 原工杰；

【导师】 丁寅；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2010， 博士

【摘要】 绝经后骨质疏松症(osteoporosis, OP)是老年妇女的常见病,多发病。雌孕激素的缺乏被认为是绝经后骨质疏松的主要原因,而骨质疏松被认为是牙周病及牙齿缺失的原因之一。临床研究显示,牙槽骨高度及骨密度的降低与雌激素水平的降低有关。与雌激素相似,孕激素的水平在妇女绝经后也显著下降。孕酮具有重要的骨保护作用,以往的研究多集中于孕酮对全身骨骼系统的影响,而关于孕酮在牙槽骨改建中的作用及机制的研究甚少。牙周膜(periodontal ligament cells,PDL)是牙骨质和牙槽骨之间的非矿化结缔组织。牙周膜能维持骨形成和骨吸收的平衡,在维持牙周组织内环境稳定中起重要的作用。因此,牙周膜是牙齿萌出和正畸牙移动的基础。人牙周膜细胞(human periodontal ligament cells,hPDLCs)来源于中胚层,它被认为是成骨细胞的来源且具有多向分化潜能。孕酮是骨代谢的调节因子,它对牙周组组织也产生潜在的影响。孕酮水平的失衡也常常导致牙周疾病的发生。但hPDLCs是否表达孕激素受体(Progesterone receptor, PR),孕酮是否能影响hPDLCs的增殖及成骨分化仍不清楚。本实验主要是从基因和蛋白水平检测PR的表达,研究孕酮在hPDLCs增殖及成骨分化过程中的作用,进而为探讨孕激素及PR在牙槽骨吸收与改建中的作用机理奠定基础。我们设计了如下实验:1.人牙周膜细胞中孕激素受体的表达取正畸拔除的健康前磨牙的牙周膜组织,进行hPDLCs的原代培养,用免疫细胞化学的方法检测PR蛋白的表达,RT-PCR的方法检测PR mRNA的表达。2.孕酮对人牙周膜细胞增殖能力的影响用含有不同浓度(1,10,100 nM)孕酮的培养液培养hPDLCs 1,2,3,4,5,6,7天,采用MTT的方法检测在孕酮作用下hPDLCs生长曲线的变化情况。用100 nM孕酮培养液培养hPDLCs3天,以流式细胞周期分析的方法检测孕酮作用下细胞周期的变化。分别以未作处理的hPDLCs作为空白对照组,特异性的PR拮抗剂RU486(100 nM)处理的hPDLCs为抑制剂组。3.孕酮对人牙周膜细胞成骨作用的影响我们研究了普通培养液和成骨诱导培养液中孕酮作用于细胞后碱性磷酸酶(Alkaline phosphatase,ALP)染色及茜素红染色钙化结节面积的变化。为了进一步研究孕酮在hPDLCs成骨分化中的作用,我们用孕酮干预hPDLCs,检测细胞ALP活性,检测骨涎蛋白(Bone sialoprotein ,BSP)、骨钙素(Osteocalcin,OCN)及骨桥蛋白(Osteopontin, OPN)的含量,研究孕酮及PR对hPDLCs成骨分化能力的影响。4.雌孕激素联合对人牙周膜细胞增殖分化的影响用雌二醇,孕酮及雌孕激素联合干预细胞。用MTT的方法检测雌孕激素对hPDLCs增殖的影响,用ALP活性分析和茜素红染色的方法检测雌孕激素对细胞成骨分化作用的影响。5.孕酮对人牙周膜细胞c-fos、c-jun和Runx2基因表达的影响用孕酮及抑制剂刺激hPDLCs,检测孕酮对细胞c-fos、c-jun和Runx2基因表达的影响。研究结果发现:1、免疫细胞化学结果显示,PR在hPDLCs的胞质和胞核中均有表达,且在胞核的表达明显高于胞质。RT-PCR结果显示hPDLCs中有PRmRNA的表达。2、孕酮干预hPDLCs后,细胞的增殖高于对照组,PR抑制剂能明显抑制hPDLCs的生长。流式细胞周期分析结果显示:与对照组相比,孕酮组S+G2M期的百分比显著增加。抑制剂组的S+G2M期的百分比低于孕酮组,有显著差异。3、在普通培养液中,孕酮组的ALP染色阳性面积和茜素红染色钙化结节面积明显高于对照组和抑制剂组。在成骨诱导液中,孕酮同样有促进成骨的作用。与成骨诱导组相比,诱导孕酮组的ALP染色阳性面积和茜素红染色钙化结节面积进一步增加。孕酮干预细胞后,hPDLCs中的ALP活性,BSP,OCN和OPN mRNA的表达升高具有时间及剂量依赖性,抑制剂组各指标减小。4、在MTT分析中,对照组的hPDLCs增殖明显低于孕酮组、雌二醇组和雌孕联合组。但孕酮组、雌二醇组和雌孕联合组之间没有显著差异。茜素红染色结果显示,雌二醇组钙化结节面积高于孕酮组的钙化结节面积,两者的面积明显低于雌孕联合组。诱导雌孕组的钙化结节面积高于诱导孕酮组和诱导雌二醇组。雌孕联合组的ALP活性明显高于孕酮组和雌二醇组。5、孕酮组c-fos、c-jun和Runx2 mRNA的表达明显高于对照组,而抑制剂组c-fos、c-jun和Runx2的表达显著降低。得出了如下结论:1、免疫细胞化学法和RT-PCR分别从蛋白水平和mRNA水平证实了PR在hPDLCs中有表达。2、孕酮能够促进hPDLCs的增殖,孕激素受体拮抗剂能有效抑制hPDLCs的增殖。3、孕酮能够促进hPDLCs的成骨分化,孕酮的作用能被孕激素受体抑制剂所拮抗。提示在hPDLCs的增殖和成骨分化中,孕酮是通过配体-受体途径来发挥其生物学效应的。4、与空白对照组相比,孕酮、雌二醇及雌孕联合应用均能促进hPDLCs的增殖及成骨分化。雌孕激素联合应用与单独应用相比,对细胞增殖的影响没有显著差异。雌孕激素联合应用比单独应用能够进一步促进细胞的成骨分化。5、孕酮能够上调hPDLCs中的c-fos、c-jun和Runx2 mRNA的表达。提示孕酮可能通过上调c-fos、c-jun和Runx2的表达促进hPDLCs的增殖和成骨分化,促进骨形成。更多还原

【Abstract】 Postmenopausal osteoporosis (osteoporosis, OP) is a common and frequently-occurring disease for older women. Estrogen and progesterone deficiencies are well-known major contributors of postmenopausal osteoporosis development. Osteoporosis is regarded as one of the causes of periodontal disease and tooth loss. Clinical observations in postmenopausal women show that a higher frequency of loss of alveolar bone height as well as a low mandible bone density is accompanied by lower estrogen level. Similar to estrogen, the serum level of progesterone also reduces markedly after menopause. Recent experimental and clinical data have also demonstrated that progesterone plays a major role in protecting the skeleton. However, less information is available on the anabolic effects of progesterone on the remodeling of alveolar bone.Periodontal ligament (PDL) is non-mineralized connective tissue located between the alveolar bone and cementum. PDL plays a vital role in maintaining homeostasis of periodontal tissues by affecting coordinated balance between bone formation and bone resorption activity. Thus, PDL is the fundamental requirement for both tooth eruption and orthodontic tooth movement. The periodontal ligament cells (PDLCs) are considered as multipotential cells and a source of osteoblasts.As well as being the regulator of the integrity of the skeleton, progesterone has the potential effects on periodontal tissues. It is obvious that periodontal disease manifestations occur when an imbalance of progesterone takes place. However, it is still not clear whether progesterone receptor (PR) is expressed in hPDLCs and whether progesterone has effects on the proliferation and differentiation of hPDLCs. Therefore, the purpose of the present study is to detect the expression of PR in hPDLCs and explore the effects of progesterone on the proliferation and differentiation of hPDLCs in vitro. This research might lay the foundation for studying progesterone and the PR mechanism in the alveolar bone resorption and remodeling.We designed and carried out the experiment as follows:1. The expression of progesterone receptor in human periodontal ligament cellsThe hPDL tissue was obtained from the extracted healthy premolar for orthodontic reasons. We performed the primary culture of hPDLCs. The PR expression in hPDLCs was detected by immunocytochemistry and the reverse transcriptase polymerase chain reaction.2. The effect of progesterone on the proliferation of human periodontal ligament cellsThe hPDLCs were stimulated with progesterone (1, 10, 100 nM) for 1, 2, 3, 4, 5, 6, and 7 days. MTT assay was performed to assess the effects of progesterone on cell growth curve. Cell cycle analysis was performed to further test the effect of 100 nM progesterone on the proliferation rate of hPDLCs by means of flow cytometry on the third day. The untreated hPDLCs served as control group. We employed RU486, a specific progesterone receptor antagonist, as antagonist group.3. The effect of progesterone on the differentiation of human periodontal ligament cellsTo quantify the effects of progesterone on osteogenic differentiation of hPDLCs, alizarin red and ALP staining were performed in basic culture medium and osteogenic medium. In this experiment we also examined the effect of the progesterone on ALP activity and the expression of BSP, OCN, and OPN mRNA in hPDLCs, respectively.4. The effects of combined estrogen and progesterone on proliferation and differentiation of human periodontal ligament cells.Cells were cultured in basic culture medium supplemented with progesterone, 17β-estrodiol or progesterone plus 17β-estrodiol. The effects of hormones on the proliferation were determined by MTT assay, and its effects on the differentiation were determined by alizarin red staining and ALP activity assay.5. The effect of progesterone on the expression of c-fos、c-jun and Runx2 in human periodontal ligament cells.Cells were untreated or treated with 100 nM progesterone in the absence or presence of 100 nM RU486.The expression of c-fos, c-jun mRNA were determined by RT-PCR. The effect of the progesterone on the expression of Runx2 mRNA in hPDLCs was determined by Realtime RT-PCR.We found that:1. In this study we proved the presence of PR mRNA in hPDLCs by using the RT-PCR and PR protein expression in hPDLCs by immunocytochemistry. The positive signal was strongly expressed in the nuclei but weaker in cytoplasm of hPDLCs.2. Progesterone can promote the proliferation of hPDLCs. When RU486 was added, this action was blocked. The percentage of the cells in S + G2M phases was higher in progesterone-treated group than that in control group and progesterone + RU486 group.3. In normal medium, the obvious mineralized nodules were detected in all the groups. The results showed that there were a few mineralized nodules in control group and progesterone + RU486 group. The amount of mineralized matrix increased in the progesterone-treated group. In osteogenic medium, increasing alizarin red–positive area was detected in osteogenic group, but the alizarin red–positive area was obviously less than that in osteogenic + progesterone group. The alizarin red–positive area was obviously decreased in osteogenic + progesterone + RU486 group. We got the similar result in ALP staining. The data have indicated that the progesterone can promote the ALP activity and the expression of BSP, OCN, and OPN mRNA in time- and dose-dependent manner in hPDLCs.4. The proliferation of hPDLCs in control group was still lower than that in progesterone-treated group, estrodiol-treated group and estrogen- progesterone group. The difference among the groups treated with progesterone, estrodiol and estrodiol-progesterone is not significant. The alizarin red-positive area increased in the progesterone-estrogen group compared with progesterone-treated group and estrogen group, and the alizarin red-positive area was obviously increased in osteogenic + estrogen + progesterone group than that in osteogenic + estrogen and osteogenic + progesterone group. The results of ALP activity assay showed that the ALP activity was higher in estrogen-progesterone group compared with other three groups.5. The results showed that the expression of c-fos, c-jun, and Runx2 mRNA was markedly enhanced in 100 nM Progesterone-treated group compared with control group, but decreased in progesterone + RU486 group.In conclusion:1. Our results demonstrate that the PR is expressed in hPDLCs at both mRNA and protein level.2. Progesterone can stimulate the proliferation of the hPDLCs, and RU486 can block the effect of progesterone.3. Progesterone can stimulate the differentiation of the hPDLCs, and RU486 can block the effect of progesterone. This also strongly supports the view that progesterone effects are mediated by the PR.4. Estrogen and progesterone also can promote the proliferation and differentiation of hPDLCs. The effect of using estrogen and progesterone together on cell proliferation is not significantly different, compared with using estrogen and progesterone alone. Moreover, combined estrogen and progesterone can further promote the differentiation of hPDLCs compared with progesterone group and estrogen group.5. Progesterone also can upregulate the expression of c-fos,c-jun, and Runx2 mRNA. Our current study suggested that progesterone may exert its bone-sparing functions by increasing the expression level of c-fos,c-jun, and Runx2 mRNA via PR in hPDLCs.更多还原