【作者】 张璇；

【导师】 丁寅；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2009， 博士

【摘要】 牙周病是一类涉及细菌微生物侵犯和宿主反应等因素影响的常见疾病。慢性牙周炎为其中最多见的一种:是由牙菌斑引起的发生在牙齿支持组织的慢性感染性炎症,通常可导致牙周组织结构破坏,牙周附着丧失,牙槽骨吸收,引起牙齿松动脱落,是成人失牙的主要原因。大量研究表明,细菌是牙周炎的始动因素,但细菌的作用会因宿主反应的不同而改变,因此认为罹患牙周炎的个体涉及遗传易感因素。目前有关基因易感因素与牙周炎相关性的研究主要采用候选基因多态性的关联研究。研究较多的牙周炎易感基因包括白细胞介素-1 （IL -1）,维生素D受体（VDR）,雌激素受体（ER）,IgG₂ Fc受体（FcγR）,基质金属蛋白酶家族（MMPs）等。由于雌激素调节骨代谢,ER被认为是骨相关疾病的重要候选基因。ER的碱基突变会引起ER的多态性,形成不同的基因型;不同的基因型会导致不同个体中ER的转录表达水平和受体功能存在差异;ER的不同基因型有可能作为基因背景,决定不同人群、不同人种对于某些疾病具有不同的易感性和不同的临床表征。迄今为止,还少有关于ER多态性和牙周炎相关性的报道。因此,我们设计了第一部分实验:雌激素受体基因多态性与慢性牙周炎相关性的研究研究样本为109例慢性牙周炎（Chronic periodontitis, CP）患者和99例牙周健康志愿者。Chelex-100法从颊粘膜拭子脱落细胞中提取基因组DNA,采用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）分析法分别检测样本人群ER-α和ER-β基因多态性;分析病例组与对照组中各基因型分布和等位基因频率。结果:Chelex-100法提取的基因组DNA可以满足本实验候选基因ER的基因多态性检测;慢性牙周炎组与正常对照组在ER-αXbaI基因型分布上有统计学差异（p=0.01）,慢性牙周炎组XX型基因频率明显高于正常对照组,此差异在女性患者中更加显著（p=0.01）,在男性患者中未见明显不同;ER-αPvuII、ER-βRsaI和ER-βAluI基因型分布在患者组与对照组中未见明显差异（p>0.05）。多元回归分析显示CP易感性与年龄、XbaI多态性相关（p<0.05）;与吸烟和PvuII、RsaI、AluI基因多态性无明显相关性。结论:慢性牙周炎易感性与雌激素XbaI基因型分布相关,在汉族女性群体中ER-α受体XX基因型可能为慢性牙周炎的遗传易感因子。牙周炎和骨质疏松是中老年妇女的常见病和多发病,尤其在绝经后妇女中有很高的患病率,并且这两种疾病存在许多共同危险因素,其中宿主因素是现今研究的热点。雌激素缺乏与牙周炎和骨质疏松这两种疾病的发生有密切关系,同时牙周炎和骨质疏松之间也存在相关性。随着女性进入绝经期,卵巢功能逐渐衰退直至丧失,体内雌激素水平日渐下降,使得妇女对骨质疏松症的易感性增加,失牙风险增高。牙槽骨的丧失,是牙周炎的一个特征性改变,主要是由于细菌感染,宿主免疫系统对感染的一种反应所致。骨量减少和骨质疏松已被认为是影响牙槽骨吸收的附加因素。学者们普遍认为,雌激素缺乏所导致的绝经后骨质疏松（post-menopausal osteoporosis, PMO）是牙周炎的易感因素之一。为了解雌激素受体多态性与全身骨密度在慢性牙周炎妇女群体中是否存在相关关系,探寻雌激素受体多态性、骨密度和牙周炎三者间的内在联系和可能的分子机制。我们设计了第二部分实验:雌激素受体基因多态性与骨密度的相关研究本研究纳入145例女性慢性牙周炎患者（绝经后女性:65例;绝经前女性:80例）和60例绝经后牙周健康女性对照人群,收集受试者的颊拭子,采用Chelex-100方法提取基因组DNA, PCR-RFLP技术检测ER-α受体的基因型分布。牙周检查包括临床附着丧失和牙周袋探诊深度。应用双能X线骨密度仪测定205例实验人群腰椎、股骨颈等部位的骨密度（bone mineral density, BMD）。统计分析组内和组间基因型与BMD的相关性。结果:未发现绝经后慢性牙周炎妇女与对照组各部位骨密度有差异;绝经前和绝经后慢性牙周炎妇女ER-α受体基因PvuII位点和XbaI位点的六种基因型与各部位骨密度间均未见有统计学差异（p﹥0.05）;但慢性牙周炎妇女绝经前后骨密度在Ward’s三角和腰椎L2-L4这两个部位有显著的统计学差异（p= 0.027和0.004）。结论:在绝经前和绝经后慢性牙周炎女性群体组内均未发现ER-α基因多态性与骨密度有相关性;但Ward’s三角和腰椎L2-L4两个部位骨密度在绝经前与绝经后的慢性牙周炎妇女组间存在差异。更多还原

【Abstract】 Periodontal disease, involving microbial encroachment and host responses, are one group of the most common disorders. Chronic periodontitis (CP), is the most one of periodontal disease. It is the inflammatory response to bacteria leads to the destruction of supporting tissue of teeth, characterized by loss of connective tissue and alveolar bone, after that, tooth mobility and abscess may occur, eventually leading to tooth loss. Although bacteria are an initial factor for periodontitis, their impact may be modified by an individual’s predisposition. It has been suggested that there is genetic basis and a predisposition for individuals to suffer from CP.Nowadays, most of the association studies have utilized the candidate gene polymorphisms approach to identify the gene variation related to periodontitis. In the last decade a multitude of research projects have been performed to investigate the association between periodontitis and polymorphisms of some genes,including pro- and anti-inflammatory cytokines, innate immune recognition receptor, and some bone metabolism related factors (such as the Vitamin D receptor, MMPs, ER, FcγR, IL-1).Estrogen regulates bone metabolism and triggers a broad array of tissue- and organ- specific physiological responses by binding to a nuclear receptor protein, the ER-αand ER-β. ER is considered the important candidate gene for bone-related diseases. The basic group mutation of ER would cause polymorphisms, formed different genotypes; different genotypes may cause difference of the transcription, or the level of expression or the function of ER receptor. To date, there is a few studies on ER polymorphisms and periodontitis. Therefore, we designed part one of the study.Part one: The association between estrogen receptor and chronic periodontitisA total of 208 unrelated Chinese individuals were selected for the study population, consisting of 109 chronic periodontitis patients and 99 periodontally healthy volunteers serving as the control group. Genomic DNA was extracted from oral mucosa swab sample of each subject by the Chelex-100 method. Determination of the ER polymorphisms was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) with XbaI and PvuII (for ER-α), RsaI and AluI (for ER-β), respectively. The distribution of genotypes and the frequency of alleles were analyzed.Results: The extraction of DNA from swab sample using Chelex-100 is more efficient and rapid than conventional phenol-chloroform extraction for studing genetic polymorphism. And DNA was satisfied to do the further PCR-RFLP experiment. The analysis of the XbaI polymorphisms in ER-αgene revealed significant differences between patients and controls. The detection frequency of XX genotype was significantly higher in the chronic periodontitis patients than in control subjects (22.02% vs. 6.06%, p=0.01). The difference between the female chronic periodontitis patients and healthy controls (29.82% vs. 3.92%, p =0.01) was statistically significant, but no difference was found between the male patients and controls (p = 0.08). Nevertheless, no significant association was noted in the frequency of both RsaI and AluI polymorphisms in ER-βgene and chronic periodontitis. Logistic regression analysis shows that the susceptivity of chronic periodontitis is correlated to age and XbaI gene polymorphisms (p<0.05), but not association with sex, smoking and PvuII, RsaI, AluI gene polymorfisms (p>0.05).Conclusions: It is indicated that the ER-αXX genotype may be a susceptible indicator for chronic periodontitis in female Han Chinese subjects.Osteoporosis is a systemic disorder of decreased skeletal mass and disturbed skeletal architecture and function that result in an increased risk of bone fractures with resultant increased morbidity and mortality. The pathogenesis of osteoporosis is complex and is believed to be an interaction between genetic susceptibility and multiple environmental risk factors, although a number of factors, including hormonal, environmental, and nutritional factors, have been shown to affect BMD. Twin and family studies have revealed that at last 40%-70% of the variation of BMD is accounted for by genetic factors. Periodontitis and Osteoporosis are common disease in post-menopausal women. There are co-risk factors in the two disorders, and the host factor is the current focus. As we known Estrogens play a significant role in bone physiology. Insufficient estrogen is believed to be one of the major causes of postmenopausal osteoporosis (PMO). 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. Scholars generally considered that PMO caused by insufficient estrogen is one of the risk factors for periodontitis. Part two: The relationship between the estrogen receptor-αgene polymorphisms and bone mineral density in periodontitis.A total of 145 unrelated Chinese female individuals were selected for the study population, consisting of 65 post-, 80 pre-menopausal female chronic periodontitis patients, and 60 post-menopausal healthy volunteers serving as the control group. Genomic DNA was extracted from oral mucosa swab sample of each subject by the Chelex-100 method. Determination of the ER-αpolymorphisms was performed by PCR-RFLP technique with XbaI and PvuII enzyme. The distribution of genotypes was analyzed. Do dental measures included Clinical attachment loss (CAL), probing pocket depth (PPD).BMD was measured at the spine, femur neck, Ward’s triangle and trochanter by dual-energy x-ray absorptiometry (DEXA).statistical analysis the relationship between genotypes and BMD.Results: There was no significant differences between ER-αPvuII, ER-αXbaI genotypes and spine, femur neck, Ward’s triangle and trochanter BMD in Post-menopausal CP patients; Meanwhile, there was no significant differences between ER-αPvuII, ER-αXbaI genotypes and spine, femur neck, Ward’s triangle and trochanter BMD in Pre-menopausal CP patients(p>0.05); However, there was association between pre- and post- menopausal CP patients (p= 0.027) at Spine L2-L4 BMD; Moreover, there was association between pre- and post- menopausal CP patients (p= 0.004) at Ward’s BMD.更多还原