【作者】 陈栋；

【导师】 吴织芬；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2007， 博士

【摘要】 牙周病是一类涉及到细菌微生物侵犯和宿主反应等因素影响的常见病之一;细菌是牙周炎的始动因素,但是细菌的作用可能会被宿主反应不同所改变。侵袭性牙周炎是常发生在全身健康的青少年和年轻成年人、以牙周支持组织快速降解破坏为特征的一类牙周炎;大量的遗传学研究认为罹患侵袭性牙周炎的个体涉及遗传易感因素。迄今大多数遗传基因易感因素和牙周病相关性研究主要采用候选基因多态性(polymorphisms)的关联研究;已有研究多关注免疫系统和炎性因子的基因,包括白介素-1(IL-1),人类白细胞抗原(HLA),维生素D受体,Fcγ受体等基因;实际上炎性因子刺激宿主细胞产生过量的基质金属蛋白酶(matrix metalloproteinases, MMPs),最终导致了牙周组织的降解和破坏。MMPs家族是由20多种锌离子依赖性内切酶组成,在胚胎发育、炎症反应和肿瘤侵袭转移等中发挥重要作用;尤其在炎症反应中加速白细胞募集、细胞因子释放和促进基质降解改建。明胶酶是MMPs家族的主要成员,包括MMP-2(明胶酶A)和MMP-9(明胶酶B);它们作用底物广泛,包括IV型胶原、明胶、弹性蛋白等。MMP-2主要由成纤维细胞、内皮细胞和破骨细胞表达;MMP-9主要由中性粒细胞、巨噬细胞和单核细胞产生。MMPs的表达活化受到转录调控、底物特异性、内源性抑制剂—组织金属蛋白酶抑制剂(tissue inhibitors of metalloproteinases,TIMPs)三方面的平衡调节。大量临床研究证实在牙周炎活跃期的龈沟液、牙龈中包括MMP-2、MMP-9在内的MMPs水平明显升高,MMPs/TIMPs比例失衡。近年来国内外学者发现在不同种族中MMP-1启动子区的单核苷酸多态性(single nucleotide polymorphism,SNP)与慢性牙周炎或侵袭性牙周炎易感性有一定相关性。促使人们把目光投向非炎症系统的MMPs家族基因作为候选基因,探寻其基因多态性和牙周炎的关系。近来在MMP-2基因启动子区发现-1306bp位点胞嘧啶替换为胸腺嘧啶(C→T)形成SNP,改变了CCACC盒而影响转录水平;在MMP-9启动子区-1562bp位点C→T产生的SNP对转录也产生显著影响,T等位基因表达水平升高;TIMP-2基因启动子区-418bp位点上鸟嘌呤转换为胞嘧啶(G→C),影响到Sp1结合位点导致TIMP-2基因转录水平的下调,进而可能影响到TIMP-2和MMP-2之间的平衡。本课题将对MMP-2,MMP-9和TIMP-2启动子区基因多态性与侵袭性牙周炎相关性进行研究。牙周专科电子病历是对牙周病临床病例样本进行牙周临床指标统计分析的理想工具,对牙周临床科研工作的开展起着不可替代的作用。随着牙周病学研究的快速发展和口腔数字化管理的普及,亟需牙周专科电子病历,加快国内牙周电子病历的研制开发势在必行。为此本课题开发构建了牙周专科电子病历。本课题还利用临床搜集的AgP患者牙龈组织标本,应用免疫组织化学法对MMP-9、TIMP-2的表达进行定位及半定量分析,探讨了MMP-9、TIMP-2在侵袭性牙周炎不同基因型牙龈组织中的表达情况。第一部分牙周电子病历的构建本研究采用微软IIS(Internet Information Server)的ASP(Active Server Page)服务器脚本语言和ACCESS数据库,实现了牙周临床指标数据录入统计和牙周健康状况全景形象展示,较为全面细致地反映患者牙周状况,可以满足牙周专科和本课题临床病例牙周指数统计的需要,为发展国内牙周专科电子病历做出有益探索。第二部分MMP-2,MMP-9和TIMP-2启动子区基因多态性与侵袭性牙周炎相关性研究研究样本为79名广泛型侵袭性牙周炎(generalized aggressive periodontitis,G-AgP)患者和128名临床牙周健康对照者;本研究样本的检验效能为75%,样本量大小符合检测效能的要求。Chelex-100法从颊拭子脱落细胞中提取DNA,采用聚合酶链反应-变性高效液相色谱法(denaturing high- performance liquid chromatography,DHPLC)和限制性片段长度多态性分析(restriction fragment length polymorphism , RFLP)法分别检测样本MMP-2-1306bpC/T,MMP-9-1562 C/T和TIMP-2-418G/C启动子区基因多态性;分析两组中基因型分布、等位基因频率,以及MMP-2和TIMP-2复合基因单体型。结果:Chelex-100法所提取DNA完全可以满足本实验候选基因的SNP检测;G-AgP组和对照组按照年龄和性别t检验p = 0.157和0.124,两组的性别、年龄匹配。经卡方检验两组中MMP-2,-9,和TIMP-2基因型符合哈代-温伯格平衡(Hardy-Wenberg equilibrium)(χ2= 0.001~0.83,p>0.05)。卡方检验均经过耶茨校正(Yates’correction),多重卡方检验校正p<0.017为有显著性意义。MMP-2-1306C/T、MMP-9-1562C/T启动子区基因多态性在G-AgP组和对照组中最常见的基因型为CC,而TT基因型少见,各基因型在两组的分布没有差异(p>0.05)。等位基因频率在两组中也没有显著性差异(p=0.707,0.858)。TIMP-2启动子区基因多态性在G-AgP组和对照组中基因型和等位基因频率的分布:在G-AgP组GC+CC基因型(48.1%)比对照组(32.0%)明显增多[p = 0.030; OR: 1.97, 95% CI: (1.11;3.50)],这种差异经过多重卡方检验校正后失去显著性意义(p>0.017);TIMP-2-418G和-418C等位基因频率在G-AgP组分别为71.5%和28.5%,而在对照组分别为82.4%和17.6%,等位基因-418C在G-AgP组的检出频率显著高于对照组[p=0.013; OR: 1.87, 95% CI: (1.17;2.99)]。MMP-2和TIMP-2复合基因单体型分析以常见的MMP-2-1306 CC+TIMP-2-418GG基因型作参照,两组中复合基因型分布差异没有显著性意义(p>0.05),即没有显示出MMP-2和TIMP-2复合基因的叠加作用和我国汉族人群侵袭性牙周炎有相关性。结论:MMP-2-1306C/T、MMP-9-1562C/T启动子区基因多态性可能不是我国汉族人群侵袭性牙周炎的遗传易感因素;TIMP-2-418G/C启动子区基因多态性与我国汉族人群广泛型侵袭性牙周炎有相关性,TIMP-2-418C等位基因可能是我国汉族人群广泛型侵袭性牙周炎的遗传易感因素。第三部分MMP-9, TIMP-2在不同基因型侵袭性牙周炎患者牙龈组织中的表达本部分对6例健康牙龈标本和9例AgP患者牙龈标本,应用免疫组织化学染色法,按照MMP-9-1562bpC/T、TIMP-2-418G/C不同基因型分组,观察牙龈组织中MMP-9、TIMP-2的表达,目的在于分析MMP-9、TIMP-2在侵袭性牙周炎不同基因型的龈组织表达情况。结果:在正常牙龈标本中MMP-9和TIMP-2几乎不表达,而在侵袭性牙周炎患者的牙龈组织中MMP-9和TIMP-2表达明显。MMP-9主要在固有层炎性细胞密集区阳性表达;TIMP-2多在上皮层表达。在病例组,含有MMP-9-1562T等位基因和不含MMP-9-1562T等位基因的牙龈组织中MMP-9表达阳性细胞数量和平均灰度值无显著差别;不含TIMP-2-418C等位基因的牙龈组织中TIMP-2表达阳性细胞平均灰度值虽然高于含有TIMP-2-418C等位基因的牙龈组织,但无统计学意义;两种基因型的TIMP-2表达阳性细胞数量统计也无显著性差异。结论:MMP-9和TIMP-2参与了侵袭性牙周炎牙龈组织的病变,但没有发现不同基因型AgP牙龈组织中MMP-9和TIMP-2表达水平有相应的变化。更多还原

【Abstract】 Periodontal diseases, involving microbial challenge and host responses, are one group of the most common disorders. Although bacteria are an initial factor for human periodontitis, their impact may be modified by an individual’s predisposition. Generalized aggressive periodontitis (G-AgP), as a subgroup of periodontitis, is characterized by rapid degradation and destruction of periodontal supporting tissue in otherwise clinically healthy the juvenile or early adult subjects. AgP is less common than chronic periodontitis (CP) and differ in many respects with regard to their etiology and pathogenesis. It has been suggested that there is a genetic basis and a predisposition for individuals to suffer from AgP.Most of the association studies to date have utilized the candidate gene polymorphisms approach to identify the gene variation related to periodontitis. Previous studies have focused on the relationship between genetic variation in the candidate genes of the immune and pro-inflammatory systems and periodontitis. These genes include interleukin-1, human leukocyte antigen, vitamine D receptor, and Fc gamma receptor et al.. Actually, these pro-inflammatory cytokines stimulate cells of the host to produce a number of matrix metalloproteinases (MMPs), which are eventually responsible for degradation of periodontal connective tissues in the pathogenesis of periodontitis. MMPs are a family consisting of over 20 related zinc-dependent endopeptidases, most of which have been demonstrated to be associated significantly with many biological or pathological processes, such as embryogenesis, inflammation, and cancer metastasis et al.. Furthermore, MMPs perform multiple roles in the host response to the procession of infection, facilitating leucocyte recruitment, cytokine and chemokine processing and matrix remodeling. Both resident cells and infiltrating inflammatory cells produce excessive MMPs that are thought to be mediators in pathogeneses of periodontitis. The important members of MMPs exampled by MMP-2 and MMP-9 (named as gelatinase A and gelatinase B), mainly cleaving type IV collagen and gelatins, are also believed to play important roles in tissue destruction in periodontitis. MMP-2 is synthesized by various fibroblasts, endothelial cells and osteoblasts, while MMP-9 is expressed by polymorphonuclear leukocytes (PMN), macrophages, and epithelial cells. Besides transcriptional regulation and substrate specificity, the activity of MMPs is also regulated by endogenous factors, including a family of anti-proteinases known as tissue inhibitors of metalloproteinases (TIMPs). Elevated MMP-2 and MMP-9 levels of tissue or gingival crevicular fluid (GCF) have been observed in inflammatory sites in periodontitis.Several studies so far focus on the association between MMP1 polymorphism and chronic periodontitis (CP) or AgP among MMPs. These results demonstrate that a single nucleotide polymorphism in the MMP1 promoter region of -1607bp is associated with generalized aggressive periodontitis (G-AgP) in the Chinese population, with severe CP in the Brazilian population and slightly with CP in the Czech population. As non-inflammatory mediators, MMPs are highlightened in the candidate gene polymorphisms related to aggressive periodontitis. Several functional single nucleotide polymorphisms in the MMP-2 and MMP-9 promoter region have distinct effects on MMP expression, which might enhance susceptibility to periodontitis. A cytosine (C) to thymine (T) substitution at position -1306bp of MMP-2 promoter region changes cis-regulatory elements by disrupting a Sp1-type promoter site (CCACC box). This functional polymorphism displays a strikingly different promoter activity between the C and T allele. Other studies indicate that the position -1562bp in the MMP-9 promoter region also has a C to T substitution, which has an allele-specific effect on MMP-9 transcription. In vitro experiments using the reporter assay technique have showed that the -1562T allele has higher promoter activity in driving gene expression than the -1562 C allele. Of the four members in the TIMP family, TIMP-2 is particularly interesting because of its dual functions in terms of regulating MMP-2 activity. A guanine (G) to C transition located at -418bp has also been identified in the consensus sequence for the Sp1-binding site in the promoter region of TIMP-2. It is reasonable to postulate that the polymorphism may down-regulate TIMP-2 expression and consequently cause an imbalance between the activities of TIMP-2 and MMP-2, which is believed to have a significant impact on periodontitis development and progression. Further research is therefore needed on the relationship between MMP-2, -9, TIMP-2 polymorphisms and AgP.Compute-based Patients Record (CPR), which is used for department of periodontology, is the perfect tool for the statistical analysis of periodontal indices in the research of clinical samples. It is indispensable for CPR to develop the research of clinical periodontology. Due to the development of periodontology and the digital management, the utilization and criteria of CPR need to be further completed and improved. In the present study, we established the new model of patients’record based on computer in stead of traditional periodontal record.Additionally, healthy and diseased gingival sample were collected from the patients with AgP or the the controls. Immunohistochemical method was used to analysis the localization and semi-quantitive expression of MMP-9 and TIMP-2. We aimed for understanding the level of expression in gingival specimen with different genotypes by the result of Part two.As mentioned above, the goal of the present study was to evaluate the genotype distribution and allele frequencies of the MMP-2,9 and TIMP-2 of G-AgP patients in a Chinese population and also to investigate whether genetic variations of MMP-2, 9 and TIMP-2 are gene biomarkers for susceptibility to G-AgP.Part one Establishment of Compute-based Periodontal case RecordIn the present study, active server page and access of internet information server were used to process the Compute-based Patient Record of periodontal indices importing. In sequence, the whole image of periodontal condition of individual can be formed in the screen of the computer in clinical practice. The designed CPR could facilitate the daily clinincal data collection and processing at department of periodontology. To the best of our knowledge, this is the first software to record periodontal indices in China.Part two Analysis of the MMP-2, MMP-9 and TIMP-2 gene promoter polymorphisms in patients with generalized aggressive periodontitis in a Chinese populationA total of 207 unrelated Chinese individuals were selected for the study population, consisting of 79 G-AgP patients and 128 periodontally healthy volunteers serving as the control group. All participants were ethnically homogeneous Han Nationality. The power calculations performed for this study show that the power of sample size was larger than 75%, which was enough to detect association with an acceptable level of confidence. Genomic DNA was extracted from oral mucosa swab sample of the study subject by Chelex-100 method. MMP-2-1306bpC/T genotypes were determined by PCR-based denaturing high performance liquid chromatography analysis while MMP-9-1562 C/T and TIMP-2-418G/C genotypes were identified by a PCR-based restriction fragment length polymorphism. The genotypes distributions and allele frequencies of three candidate genes were evaluated by Chi-square test, so were a potential composite MMP-2 and TIMP-2 halpotypes.Results The extraction of DNA from swab sample using Chelex-100 is more efficient and rapid than conventional phenol-chloroform extraction for studying genetic polymorphism. Two groups were comparable in terms of gender and age (p = 0.157 and 0.124, respectively). The mean ages of the patients and the controls were 27.3 and 28.5 years old respectively. The distribution of MMP-2, MMP-9 and TIMP-2 genotype in the G-AgP patients and the controls was in accordance with Hardy-Weinberg equilibrium by Chi-square test (χ2 = 0.001 to 0.83, P >0.05). Chi-square test after Yates’correction was used to investigate the possible association of the genotypes with the G-AgP. Corrected p values were calculated for multiple testing by the Bonferroni method. If 3 separate comparisons had been made, then the corrected significance level would be 0.017. In the patients and the controls, the most common genotype of MMP-2 and MMP-9 gene was CC, while the TT homozygote patients were very rare. No significant difference was observed in the distributions of the genotypes and alleles in MMP-2 and MMP-9 between the patients and the controls (p>0.05). The homozygotes and heterozygote of TIMP-2 appeared to be distributed evenly in the two groups. Yates’correction showed a marked difference of the distribution of TIMP-2 GC and CC genotypes in the groups (p=0.030; OR: 1.97, 95% CI: [1.11; 3.50]), which would be no significant after adjusted p-values level for multiple comparison. The frequencies of the alleles TIMP-2-418G and -418C were 71.5%and 28.5% in the patients and 82.4% and 17.6% in the controls, respectively. A significant increase in the allele C of the patients compared to the controls occurred (p =0.013; OR: 1.87, 95% CI: [1.17; 2.99]). Because the MMP2 -1306 TT and TIMP-2-418 CC homozygotes were rare in the present study, these genotypes were respectively combined with the MMP2 -1306 CT and TIMP-2 -418 GC heterozygotes for estimation of susceptibility to G-AgP. Using the most common genotype MMP-2CC and TIMP-2GG as a reference, however, no significant synergistic effect on the occurrence of G-AgP was observed in subjects carrying the composite genotypes of MMP-2 and TIMP-2 variants (p >0.05).Conclusion Although gene polymorphisms for MMP-2 and MMP-9 did not show any association with the G-AgP, the analysis of the TIMP-2 -418G to C gene polymorphism revealed significant differences between the patients and controls. Compared with controls, a significant increasing trend of TIMP-2 -418C carrier in the G-AgP patients occurred (p=0.013). The carriers of TIMP-2 -418C polymorphism in the Chinese subjects may be more at risk of suffering from G-AgP.Part three Expressions of MMP-9 and TIMP-2 in the gingival tissues of AgP patients with different genotypes.Six healthy gingival tissues and nine AgP gingival samples were obtained in the procedure of periodontal surgery. By using immunohistochemistry and computerized image analysis, the expression level of MMP-9 and TIMP-2 was assessed on the different groups in according to MMP-9-1562bpC/T and TIMP-2-418G/C genotypes.Results Rare expression of MMP-9 and TIMP-2 was observed in the healthy gingival tissues. The distribution of MMP-9 was trended to the connective tissue nearby inflammatory cell. The TIMP-2 was often observed in epithelium layer. As to the amount and the gray value of the MMP-9 positive cells, there was no significant difference between both the groups, with or without MMP-9-1562T alleles. Similarly, no significant difference was found in the amount and the gray value of TIMP-2 positive cells between both the groups, with or without TIMP-2 -418C alleles.Conclusion MMP-9 and TIMP-2 are involved in the pathological gingiva of AgP. Aciassaton between MMP-9/TIMP-2 expression level and their respective genotypes in gingival sample can not be demonstrated.更多还原