【作者】 刘溦；

【导师】 章锦才；

【作者基本信息】 南方医科大学 ， 人体解剖学， 2011， 硕士

【摘要】 牙周病是口腔两大类主要疾病之一,在世界范围内均有较高患病率,在我国患病率更高于龋病,是成人牙齿缺失的主要原因。牙周病主要包括牙龈炎和牙周炎。慢性牙周炎(chronic periodontitis, CP)是最常见的一类牙周炎,是慢性炎症性疾病,主要临床特征有牙龈炎症、牙周附着丧失、牙槽骨吸收,严重者可出现牙齿移位和松动,最后导致牙齿缺失。尽管致病菌的存在是牙周病炎发生的必要条件,但是单有微生物尚不足以引起疾病,宿主的易感性也是基本要素。近年来大量的研究表明,与遗传有关的宿主易感性能影响和改变宿主对微生物的反应,并决定疾病的进展速度和严重程度。随着对细胞因子(Cytokine, CK)生物学效应认识的不断深入,发现调控细胞因子的基因变异能解释疾病进程中的一些个体间差异,所以近年来一些有关细胞因子基因多态性与疾病关系的研究已经有报道。而以往对于牙周炎相关的细胞因子研究热点多集中于白细胞介素-1 (interleukin-1, IL-1)、肿瘤坏死因子α(tumor necrosis factorα, TNF-α)、IgG2 Fc段受体基因、维生素D受体(vitamin D receptor, VDR)等。IL-1基因多态性是第一个被报道的与牙周炎有关的白细胞介素的基因标志。白细胞介素-4(interleukin-4, IL-4)是一种基因多效性细胞因子,它抑制Thl细胞,同时刺激Th2类型免疫反应。它同时也是一种潜在的巨嗜细胞功能下调因子,能引导单核细胞调亡。在牙周炎病变中,IL-4抑制巨嗜细胞,下调CD14受体,CD14受体是一种脂多糖的关键受体。据此可以推测,缺少IL-4对单核细胞的下调导致了牙周炎中的组织破坏。IL-4的基因多态性主要包括启动子区-590位点C/T、-34位点C/T的单核苷酸多态性(SNP),内含子2内一个70bp的重复序列多态性,它们被认为能够调节IL-4的产生。IL-4基因多态性于牙周炎的相关性在国外已有报道,但结果不尽相同。对于我国汉族人慢性牙周炎患者IL-4基因多态性分布特点如何,国内尚未见报道。研究目的：本实验的目的是研究一个中国汉族人群中白细胞介素-4基因启动子区-590C/T和-34C/T位点与基因多态性与慢性牙周炎易感性的关系。研究方法所有受试者广东省口腔医院牙周科及门诊检查患者,每位受检者符合以下条件：40岁及以上年龄、全口至少有14颗可以进行牙周评价的牙齿(至少有4颗磨牙)；中国汉族和没有亲缘关系的个体；无全身系统性疾病；无正畸治疗史；3个月内未应用抗生素；未经过系统牙周治疗；1年内未接受过洁治；妇女无妊娠及哺乳。所有受试者分为2组：慢性牙周炎组(CP)共104例：健康对照组共106例。慢性牙周炎诊断参照1999年牙周病分类国际研讨会的诊断标准。受检者均检查六颗指数牙：11、16、26、31、36、和46,如果指数牙缺失则以邻牙代替,每颗牙记录6个位点。并记录除第三磨牙外的所有缺牙数。健康对照组的纳入标准为全口牙的牙周探诊深度≤3mm,无附着丧失,龈退缩≤1mm,无探诊出血,全身健康。所有受试者记录性别、年龄、民族、吸烟状况、体重、身高、压力、系统性疾病史及牙周病史。取颊粘膜脱落细胞棉拭子,并对每位受试者进行牙周检查,助手记录,为了保证检查的准确性和一致性,本研究的检查均由经过培训和校正的同一人员完成。采用CHELEX-100法抽提基因组DNA,分光光度计检测DNA浓度。采用多聚酶链反应-限制性片段长度多态性(PCR-RFLP)法测定IL-4启动子区-590,-34位点单核苷酸多态性位点的基因型,引物按文献设计。本研究中我们随机抽取了约20%样本进行直接测序,以验证多聚酶链反应-限制性片段长度多态性(PCR-RFLP)法的正确性。本研究统计通过SPSS10.0软件完成。计量资料描述时用x±s,分析时用t检验或方差分析,每个位点在两组间的基因型分布和等位基因频率通过x2检验来完成。多因素分析采用logistic回归分析。P<0.05为有显著性差异。研究结果：比较104例慢性牙周炎患者和106例健康对照个体的基线资料,其中年龄和缺牙数有差异,P<0.05。IL-4-590位点CP组和对照组间CC、CT、TT三种基因型分布(CC=1、CT =36、TT=67;CC=4、CT=38、TT=64)没有统计学差异(z2=1.904,P=0.386),等位基因频率(C/T=38/170;C/T=46/166)也没有统计学差异(x2=0.771, P=0.380)。IL-4-34位点CP组和对照组间CC、CT、TT三种基因型分布(CC =2.CT=29、TT=73;CC=4、CT=37、TT=65)没有统计学差异(z2=2.081,P=0.353),等位基因频率(C/T=33/175;C/T=45/167)也没有统计学差异(x2=1.995,P=0.158)。因为两组样本资料在基线对比是时我们发现年龄和缺牙数两个变量在组间有差异(P=0.012；P=0.002),所以我们作了调整了年龄和缺牙数的多因素logistic回归分析,结果显示IL-4-590位点基因多态性(β=-0.279,W=1.116,P>0.05)和IL-4-34位点基因多态性(p=0.423,W=2.425,P>0.05)不是慢性牙周炎的影响因素。而缺牙数是慢性牙周炎的影响因素(β=-0.183,W=5.047,P<0.05, OR=0.833)。本研究人群中IL-4-590位点T等位基因频率为80%,与日本人、中国台湾人很接近,而明显高于高加索人群(16.2%—47.6%)。样本进行直接DNA测序的结果与多聚酶链反应—限制性片段长度多态性法的结果一致。研究结论：1.在中国汉族人群中,IL-4-590位点多态性不能独立作为慢性牙周炎的易感因素。2.在中国汉族人群中,IL-4-34位点多态性不能独立作为慢性牙周炎的易感因素。3.在IL-4启动子区单核苷酸多态性方面,中国汉族人群和高加索人群有不同的基因背景,而与日本、韩国等东亚人群有相似的基因背景。更多还原

【Abstract】 Periodontal diseases are one of the two most common oral diseases and the major cause of tooth loss in adults. In China the incidence of periodontal disease is higher than that of caries. Periodontal diseases mainly consist of gingivitis and peirodontitis. Chronic periodontitis (CP), as a kind of chronic inflammatory disease, is one type of the most commonly seen periodontitis. The main clinical manifestations of CP include gingival inflammation, periodontal attachment loss, alveolar bone absorption, tooth mobilization and dislocation, and even tooth loss in the end. Although periodontal bacteria are the causative agents in periodontitis, bacteria alone cannot cause the disease. The host susceptibility is also a fundamental factor. Studies in recent years show host susceptibility that associated with heredity can effect and alter host response to bacteria and determine the subsequent progression and severity of the disease.As the study of biological effect of cytokine (CK) goes further, studies about the relationship between cytokine gene polymorphism and diseases show gene variation regulating cytokines can explain the difference among individuals during disease progression. Previous studies about cytokines related to periodontitis are mainly focused on interleukin-1 (IL-1), tumor necrosis factor a (TNF-a), IgG2 Fc fragment receptor gene and vitamin D receptor (VDR). IL-1 gene polymorphism is the first reported interleukin genetic marker associated with periodontal diseases. Interleukin-4 (IL-4) is a pleiotropic cytokine that inhibits Thl cells while stimulating a Th2-type of immune response. It is also a potent downregulator of macrophage function and induces apoptosis in monocytes. IL-4 inhibits the persistence of macrophages in periodontitis lesions and downregulates CD 14 receptor, one of the key receptors for lipopolysaccharides. Thus, it was proposed that a lack of downregulation of monocytes by IL-4 leads to tissue destruction in periodontitis. Several polymorphisms in the IL-4 gene include a single nucleotide polymorphism (SNP) C to T in the IL-4 promoter region at position -590, one SNP at -34 C/T, and a 70bp variable number tandem repeat (VNTR) in the third intron, some of them are implicated in the regulation of IL-4 production. The correlation between IL-4 gene polymorphism and the risk for periodontitis was reported in others countries, while the findings in these studies are contradictory. There is no report on the characteristic of IL-4 gene polymorphism in Chinese chronic periodontitis patients. Hence, the aim of this study is to investigate the correlation between SNP in IL-4 promoter region at position -590 and -34 and the susceptibility to chronic periodontitis in Chinese Han nationality people..Objective:The aim of this study is to investigate the correlation of the IL-4 polymorphisms in the promoter region (-590 C to T and -34 C to T) and the susceptibility to chronic periodontitis in Chinese Han nationality people.Methods:All subjects were recruited from outpatients and inpatients referred to Guangdong Provincial Stomatological Hospital. All patients were 40 years old or greater, Chinese Han descent and genetically unrelated. Exclusion criteria were as follows:known systemic disease, history of orthodontic treatment, receiving systemic antibiotic treatment in the preceding 3 months, receiving periodontal therapy in the preceding 1 year, and pregnant or lactating females.The subjects were categorized into two groups:the chronic periodontitis group (n=104) and healthy control group (n=106). CP patients were identified based on the clinical criteria proposed by the 1999 International World Workshop for a Classification of Periodontal Disease and Conditions. The following data of the subjects were recorded:gender, age, ethnicity, smoking status, body weight, height, medical history and periodontal history. Clinical periodontal parameters of the six index teeth (11,16,26,31,36, and 46) were obtained at six sites for each examined tooth. Moreover, missing teeth were recorded except for the third molars. The criteria of healthy controls included probing pocket depth (PD) less than 3mm, no attachment loss, gingival recession less than lmm, no sites with bleed on probing (BOP), and a general healthy condition. The status of gender, age, nationality, smoking, body weight, body height, stress, medical history, and periodontal history of each participant were recorded. A buccal swab sample was obtained by twisting a swab inside each participant’s cheek. In order to keep accuracy and consistency, all periodontal examinations were carried out by a single trained and calibrated examiner.Genomic DNA was extracted from buccal swabs by CHELEX-100 method. The DNA concentration was estimated by using a spectrophotometer. Genotyping of the -590 and -34 in IL-4 promoter was performed with polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) technique. To confirm the accuracy of the PCR-RFLP,20% samples were randomly selected for direct DNA sequencing.A computer package SPSS 10.0 was used to detect significant differences between the study groups.x±s was used to describe quantitative data, while t-test or analysis of variance (ANOVA) was used to analyze quantitative data. Chi-Square (χ2) test was used to investigate genotype distribution and allele frequencies in each locus between the study groups. Logistic regression analysis was used to perform multifactor analysis. P value less than 0.05 was considered statistically significant.Results:Age and number of missing teeth showed a significant difference (P<0.05) when clinical parameters of CP and control group were compared.No significant difference (χ2=1.904, P=0.386) was found in genotype distribution (CC, CT, and TT) of IL-4 -590 between CP group and control group. No significant difference (χ2=0.771, P=0.380) was found in allele frequencies of IL-4-590 between CP group and control group. No significant difference (χ2=2.081, .P=0.353) was found in genotype distribution (CC, CT, and TT) of IL-4 -34 between CP group and control group. No significant difference (χ2=1.995,P=0.158) was found in allele frequencies of IL-4 -34 between CP group and control group.Two variables, age and number of missing teeth, were significantly different between the CP and control groups (P=0.012; P=0.002) when clinical parameters comparison was performed, so the logistic regression analysis was regulated for age and number of missing teeth. It was found both IL-4 -590 gene polymorphism (p=-0.279, W=1.116, P>0.05, OR=0.756) and IL-4 -34 gene polymorphism (β=0.423, W=2.425, P>0.05, OR=1.527) were not influential factors of CP. While the number of missing teeth was a influential factor of CP (β=-0.183, W=5.047, P<0.05, OR=0.833).The allele frequency of T in IL-4-590 were 80% in population of our study, which was close to Japanese and China Taiwanese but obviously higher than Caucasian (16.2%～47.6%).The results of direct DNA sequencing were the same with the results of PCR-RFLP.Conclusion:1. IL-4-590 C/T gene polymorphism is not a susceptible factor of chronic periodontitis in Chinese Han nationality population.2. IL-4-34 C/T gene polymorphism is not a susceptible factor of chronic periodontitis in Chinese Han nationality population.3. In terms of single nucleotide polymorphism in IL-4 promotor, Chinese Han nationality population has a different gene background compared with Caucasian, while has a similar gene background compared with east Asian population such as Japanese and Korean.更多还原