【作者】 冯二玫；

【导师】 章锦才；

【作者基本信息】 南方医科大学 ， 口腔临床医学， 2010， 硕士

【摘要】 糖尿病(DM, Diabetes Mellitus)是一种多病因的代谢性疾病,特点是慢性高血糖,伴随因胰岛素分泌及／或作用缺陷引起的糖、脂肪和蛋白质代谢紊乱。糖尿病包括两种类型1型即胰岛素依赖型(insulin-dependent-diabetes mellitus, IDDM)和2型即非胰岛素依赖型(non-insulin-dependent-diabetes mellitus, NIDDM)。目前糖尿病已经成为危害大众健康的主要疾病之一。糖尿病患者在世界范围内正在迅猛增加,预计到2030年糖尿病患者将达到3亿,糖尿病及其伴随的并发症日益受到人们重视,糖尿病可影响牙龈炎和牙周炎的发生发展：1型糖尿病儿童牙龈炎发生率显著高于非糖尿病患者,其牙龈发生炎症的位点是非糖尿病患者的两倍,有研究表明糖尿病患者牙周病发生率为75%。血糖水平影响牙周组织病变的严重程度,血糖控制不良患者牙周组织破坏更严重,且更容易出现不易愈合的牙周病损。牙周炎是由菌斑微生物引起的慢性感染性疾病,主要表现为牙龈炎症、牙槽骨吸收、附着丧失、牙松动。是成人牙缺失的主要原因。牙周炎的病因除菌斑牙石等局部刺激因素外,还受到全身健康状况如妊娠不良、心血管疾病、糖尿病等的影响。由以糖尿病对牙周组织的影响倍受关注。糖尿病与牙周炎关系密切,二者均是多因素共同作用的疾病,糖尿病本身不引起牙周炎,但由于糖尿病产生的糖代谢紊乱、微血管病变,糖激化终末产物以及糖尿病对牙菌群、中性粒细胞功能、炎症反应强度及组织愈合能力的影响,均可导致牙周微循环障碍,促进牙周炎的发生发展。研究发现糖尿病人群中,牙周病的发病率高,病变损害严重且进展迅速。2型糖尿病患者牙周炎发病率是非糖尿病患者的3倍左右。动物模型研究表明糖尿病大鼠破骨细胞数量,活性远高于正常大鼠,糖尿病可引起持续更久的炎症反应,更多的附着丧失,更严重的牙槽骨吸收,使骨修复能力明显受损,防碍新骨形成。糖尿病与牙周炎呈双向关系,一方面血糖升高是牙周炎的危险因素,另一方面牙周炎做为局部疾病对全身组织或器官产生影响,牙周感染产生的炎症因子如TNF-α进入系统循环,可引起胰岛素抵抗。研究表明牙周的非手术治疗对糖尿病患者的血糖控制有一定的影响。糖尿病患者常伴随牙槽骨吸收,研究发现糖尿病大鼠牙槽骨骨吸收活跃,骨皮质变薄,其表面成骨细胞数目减少,很少见新骨形成,2型糖尿病患者发生牙槽骨吸收的危险性是非糖尿病患者的4倍。研究发现RANKL/OPG与牙周炎牙槽骨吸收有密切联系。核因子KB受体活化因子配体(receptor activator of nuclear factor-kB ligand, RANKL)是肿瘤坏死因子配体超家族成员,来源于巨噬细胞,成纤维细胞,成骨细胞和T细胞。可结合于前体破骨细胞和破骨细胞表面,促进破骨细胞的分化成熟,诱发骨吸收。骨保护素(osteoprotegerin, OPG)是肿瘤坏死因子受体超家族成员,该蛋白与骨密度及骨含量有关,过度表达该蛋白的转基因小鼠骨质坚硬,敲除该基因的小鼠会发生严重的骨质疏松。OPG的主要作用是影响骨代谢,抑制破骨细胞的发生、分化、活化成熟及促进其凋亡。OPG是RANKL的天然抑制剂,可竞争性的抑制RANKL与核因子kB受体活化因子(receptor activator of nuclear factor-kB, RANK)的结合,从而抑制骨吸收。OPG/RANKL/RANK是破骨细胞活化的分子基础,RANKL与OPG的变化影响破骨细胞的形成和活化,从而影响骨吸收。糖尿病患者存在特异与非特异免疫功能异常,牙周炎的发生发展亦与免疫异常有关。而CD4,CD8是重要的免疫细胞,二者之间的平衡对于维持机体免疫应答起作用。本实验在没有外界刺激因素作用下,通过检测CD4,CD8在大鼠牙周组织的表达改变分析糖尿病状态下牙周组织免疫细胞功能的变化,通过检测RANKL,OPG在大鼠牙周组织中的改变情况,分析糖尿病对大鼠牙槽骨的影响。探讨糖尿病大鼠牙周组织病理改变的可能机制。目的1了解糖尿病状态下大鼠牙周组织的形态学改变；2了解糖尿病状态下大鼠牙周组织细胞免疫调节功能的改变；3通过了解牙周组织RANKL,OPG改变情况分析糖尿病大鼠牙槽骨吸收的可能风险。方法：1建立糖尿病大鼠模型SD大鼠随机分成两组：糖尿病组(DM n=30)和正常对照组(N n=8)。糖尿病大鼠高热量饲料喂养1个月后,建立肥胖模型,然后一次性静脉注射链脲佐菌素(streptozotocin,STZ) (55mg/kg),建立糖尿病模型；对照组大鼠普通饲料喂养1个月后,一次性静脉注射0.1mol/L,pH4.5相同剂量的柠檬酸缓冲液；检测血糖、尿糖改变情况。糖尿病大鼠模型成功的标准为空腹状态下(禁食8小时)血糖值≥11.2mmol/L,尿糖为+++,24小时饮水量>50ml,体重下降且这些症状持续两周。2检查牙周情况于实验开始的第16周,3%戊巴比妥钠麻醉两组大鼠,观察两组大鼠牙龈的颜色、形状、质地、有无出血及牙龈退缩情况,用牙周探针探查大鼠牙周组织附着水平。3牙周组织形态学观察按实验分组,于实验开始的16周后在戊巴比妥钠麻醉下,行腹主动脉或内眦静脉放血处死大鼠,解剖上颌骨,4%多聚甲醛溶液固定24小时(4℃),EDTA(pH=7.4)液脱钙两周,洗涤,脱水,透明,浸蜡,石蜡包埋,制成3um厚连续切片,石蜡切片常规脱蜡,脱水,HE染色,光镜视野下观察,采集图像。4大鼠牙周组织CD4,CD8的免疫组织化学糖尿病组及正常组大鼠石蜡切片脱蜡,复水,3%过氧化氢液灭活内源性过氧化物酶,枸橼酸盐缓冲液行热修复,10%山羊血清封闭,小鼠抗大鼠CD4,CD8单克隆抗体按1：100稀释,37℃恒温箱孵育1小时,加入FITC标记羊抗小鼠IgG(1：50稀释),37℃恒温箱孵育30分,激光扫描共聚焦显微镜采集图像,行灰度值分析。5大鼠牙周组织RANKL,OPG的免疫组织化学糖尿病组及正常组大鼠石蜡切片脱蜡,复水,3%过氧化氢液灭活内源性过氧化物酶,枸橼酸盐缓冲液行热修复,10%山羊血清封闭,兔抗大鼠RANKL,OPG单克隆抗体按1：100稀释,37℃恒温箱孵育1小时,加FITC标记羊抗兔IgG(1：50稀释),37℃恒温箱孵育30分钟,激光扫描共聚焦显微镜采集图像,行灰度值分析。6统计学处理应用SPSS17.0做统计分析处理,结果以X±s表示,方差齐性的样本采用独立样本t,方差不齐样本采用t’检验。p<0.05为差异有统计学意义。结果：1实验动物一般情况除糖尿病组2只大鼠死亡外,其余大鼠精神状态良好,活动自如,糖尿病组在注射STZ一周后尿糖增高,血糖值均高于11.2mmol/L,尿糖增高为+++,且出现多饮,体重下降,正常组血糖无明显变化。2牙周组织情况糖尿病组和对照组大鼠牙龈形状、质地没有明显异常,未发现牙龈萎缩,牙周探诊水平维持在初始水平,但糖尿病组大鼠牙龈颜色轻微发红,牙龈出血指数较对照组稍偏高3 HE染色观察糖尿病组大鼠牙周组织HE染色可见牙周膜纤维排列紊乱,破骨细胞形成。对照组大鼠胶原纤维排列整齐,未见破骨细胞。4牙周组织CD4,CD8荧光免疫组织染色结果糖尿病组CD4表达高于对照组(t=4.167 p=0.001),差异有统计学意义；糖尿病组CD8表达高于对照组(t=2.971 p=0.008),差异有统计学意义。5牙周组织RANKL,OPG荧光免疫组织染色结果糖尿病组RANKL表达高于对照组(t=-5.348 p=0.000),差异有统计学意义；与正常对照组相比,OPG表达降低,差异无统计学意义(t=-1.806 p=0.090)。结论：1糖尿病组大鼠HE染色出现破骨细胞,骨吸收陷窝及排列紊乱的胶原纤维,提示糖尿病虽然不能造成牙周组织实质性损害,但在一定程度上改变了牙周组织结构,影响其支持功能和防御功能,可能导致其抗感染能力下降。2糖尿病大鼠牙周组织CD4,CD8细胞表达水平高于对照组,提示糖尿病大鼠牙周组织细胞免疫功能可能出现变化,存在免疫调节功能和T细胞活化异常。3与正常对照组相比,糖尿病组大鼠RANKL表达升高,差异有统计学意义,OPG表达降低,差异无统计学意义,提示局部RANK和OPG之间出现平衡失调,破骨细胞形成增加,最终可导致牙槽骨吸收。更多还原

【Abstract】 Diabetes mellitus(DM) comprises a group of metabolic diseases characterized by hyperglycaemia resulting from defects in insulin secretion, insulin action or both. It is an evolving disease with two types:type 1 diabetes mellitus results from cellular mediated autoimmune destruction of pancreaticβ-cell, and type 2 diabetes mellitus, usually called non-insulin-dependent-diabetes mellitus, results from insulin resistance, which alter the use of endogenously produced insulin at the target cell. Now DM has been become one of the major diseases which damage public healthy and is expected to increase in prevalence to 300 million by 2030. More attention has been given to diabetes mellitus and its complications, evidence consistently reveals that diabetes is a risk factor for increased severity of gingivitis and periodontitis. The level of glycemic control appears to be an important determinant in this relationship. Diabetes patients with poor glycemic control are at great risk for progression of periodontal destruction over time, and are more likely to have severe periodontitis than those with well controlled DM.Peridontitis is a chronic infected diseases caused by bacteria, characterized by gingival inflammation, alveolar bone resorption, attachment loss. periodontitis is one of the major cause of tooth loss in adults. While association between periodontitis and several chronic systemic diseases have been demonstrated in recent years, the interaction between periodontitis and DM has been the most consistently supported.DM and periodontitis are both chronic inflammatory disorders that have a major impact on the health and well being of millions of individuals worldwide, there is a close relationship between DM and periodontitis, DM do not cause periodontitis itself, but DM can cause microcirculation disorder of periodontal tissues. A large number of investigations have provided evidence that diabetes mellitus increase the risk and severity of periodontitis. Patients with type 2 diabetes had a 3-fold increased risk of having periodontitis compared to non-diabetic subjects. Similar results have been found in animal studies, the amount and activity of osteoclast were higher in diabetic rats than normal rats. DM can cause more inflammation, more attachment loss and more alveolar bone resorption. DM and periodontitis are two-way relationship:on the one hand, hyperglycaemia is a risk factor for periodontitis, on the other, periodontitis, as a local diseases, may cause insulin resistance. Non-surgical periodontal therapy affect the metabolic control.Alveolar bone resorption is often followed to diabetes patients, patients with type 2 diabetes had a 4-fold increased risk of having alveolar bone resorption compared to non-diabetic subjiecs.It is found that RANKL/OPG have a relationship with alveolar bone resorption. Receptor activator of nuclear factor-κB ligand (RANKL)is a member of the TNF family,origins from macrophage, fibroblast, osteoblast, and T cells, it can bond to the surface of osteoclast and preosteoclast, and can induce differentiation and maturate of osteoclast and preosteoclast, induce bone resorption. Osteoprotegerin (OPG) is a member of the TNF receptor superfamily, OPG affects bone metabolism, inhibits differentiation, activity and maturate of osteoclast. OPG is a nature inhibitor to RANKL, it can competitive inhibit combination of RANKL with RANK, induce bone resorption. RANKL/RANK/OPG are molecule basement for osteoclast activity,the change of RANKL,OPG affect formation and activity of osteoclast.The abnormalities in the immune system have been considered a major factor contributing to infections in diabetic patients. The development of periodontitis also related to immnue cell-mediated immunity which is mediated by T lymphocytes, and CD4,CD8 are important immunal cells, the balance of CD4,CD8 is important to immune response.In this study, the changes of CD4,CD8 were tested in peridontal tissues of rats, and immune celluar function were analyzed in diabetes states. The expression of RANKL,OPG were tested in peridontal tissues of rats, and it was analyzed whether DM has an effects on alveolar bone of ratsObjective:1 to compare morphology changes of periodontal tissues between diabetic group and normal group.2 to compare CD4,CD8 expression changes of rats periodontal tissues between diabetic group and normal group.3 to compare RANKL,OPG expression results of rats periodontal tissues between diabetic group and normal group.Methods:1 construct models of type 2 diabetes mellitus of SD rats:SD rats were randomly divided into following groups:diabetic group(n=30) and normal group(n=8). Rats in diabetic group were foraged for establishing obese model in the first month, the diabetes was induced in by intravenous administration of streptozotocin (STZ)(55mg/kg). The normal group were breed with non-high calorie forage for one month,then were injected with the same dosage of citrate buffer sloution.The urine glucose and blood glucose were monitored. The standard of diabetes model is:blood glucose higher than 11.2mmol/L and the urine glucose was increased to+++, after fasting for 8h; the level of drinking water was less than 50ml in 24h, weight loss and then these symptoms keep for 2 weeks.2 describing periodontal tissue conditions:At the 16 week, rats were executed, observed the colour, shape, texture and the recession of the gingiva, probed the periodontal tissues with peridontal probe.3. observation periodontal tissueAt the 16week, executing rats, anatomying maxillary bone, then fixed in 4% formalin for 24h(4℃), dcalcification for two weeks, ablution, anhydration, clearing, bathing in paraffin, then paeaffin imbedding,mading section.dying with HE.4. imunohistochemistry of CD4,CD8Sections were deparaffinaged as routinely, deactivated the endogenous peroxydasse with 3% hydrogen perixide, did febrile antigen recovery with citrate buffer solution,10% goat serum was added, CD4 and CD8 monoclonal antibody of Mouse-anti-Rat were diluted according to 1:100, incubated in the attemperator at 37℃for one hour, adding FITC-Goat Anti-Mouse IgG (H+L) (diluted according to 1:50), incubated at 37℃for 30min, Using LSCM to test CD4,CD8 expression in diabetes group and normal group.5. imunohistochemistry of RANK,OPGSections were deparaffinaged as routinely, deactivated the endogenous peroxydasse with 3% hydrogen perixide, did febrile antigen recovery with citrate buffer solution,10% goat serum was added, RANKL and OPG monoclonal antibody of Rabbit-anti-Rat were diluted according to 1:100, incubated in the attemperator at 37℃for one hour, adding FITC-Goat Anti-Rabbit IgG (H+L) (diluted according to 1:50), incubated at 37℃for 30min, Using LSCM to testRANKL,OPG expression in diabetes group and normal group.6 statistic analysis: Using spss 17.0 for statistic analysis, data are expressed as mean±SD.Results:1 general conditions of experimental animalIn diabetic group besides two rats died, other rats are all in good condition. After intravenous administration of STZ in diabetic group, the urine glucose was increased to +++, the blood glucose was higher than 11.2mmol/L, and rats occurred to polydipsia and weight loss.There was no obvious changes of blood glucose in normal group.2 conditions of periodontal tissues:During the experiments, compared with normal group,there were no obvious difference in shape and texture of gingivia in diabetic group. The gingivia recession and attchment loss were not found,the depth of periodontal pocket keeped the same level as the beginning. But in debitic group, the rats gingiva colour became redder, the gingiva bleeding index was a little hingher than normal group.3.HE dyeingIn diabetic group, it occurred osteoclasts, disordered ligament fibres,these did not happened in normal group.4 results of CD4,CD8 imunohistochemistryIn diabetic group CD4 expression was higher than nrmal group,with statistically significant(p=0.001). CD8 expression was higher with statistically significant (p=0.008)5 results of RANKL,OPG imunohistochemistryThe results showed that RANKL were significantly higher than the normal group(p=0.000), compared with normal group, OPG was lower in the diabetic group, but have no significatant difference(p=0.090) Conclusion:1 in diabetic group, it occurred osteoclasts, and there were abnormality of collagen fibers, which hints that DM can change structure of periodontal tissues in some degree,and may cause decreasing of anti-infection ability in periodontal tissues.2 the results of imunohistochemistry showed that CD4,CD8 were significantly higher in diabetic group(p<0.05), it was inferred that cell immunity in periodontal tissues may be changed in the state of diabetes, and so did as immune regulation and T cell activation.3 compared with normal group, OPG was lower in the diabetic group. but there was no significatant difference(p>0.05), RANKL were significantly higher than that of the normal group(p<0.05), it hint that RANKL and OPG were imbalance in periodontal tissues, it may induce the increasing of osteoclast, even absorption of alveolar bone.更多还原