伴糖尿病牙周炎大鼠模型的建立及其颈动脉血管的病理特点

Establishment of rat model with diabetes mellitus and concomitant periodontitis and the carotid artery lesions in the model rats

目的 观察2型糖尿病（diabetes mellitus,DM）合并慢性牙周炎（chronic periodontitis,CP）的SD大鼠颈动脉血管的病理改变,探讨CP对2型DM血管病变的影响.方法 将清洁级SD雄性大鼠按随机数字表法随机分为4组,正常对照组（normal control,NC组）、DM组、CP组、DM＋CP组.DM模型采用高脂高糖饮食喂养8～10周,小剂量多次腹腔注射链脲佐菌素,动态监测血糖.CP模型采用缠绕4-0号缝线于0.2 mm正畸结扎丝上进行牙周结扎,并涂布牙龈卟啉单胞菌（Porphyromonas gingivalis,Pg）悬液.14周后每组处死7只动物,收集组织样本,颌骨样本分离牙龈后亚甲基蓝染色,观察牙周炎骨吸收情况;收集双侧颈动脉血管组织标本,左侧进行实时荧光定量PCR检测颈动脉血管壁组织的Pg,右侧行HE染色观察血管的病理变化.结果 DM及DM＋CP组大鼠在链脲佐菌素注射后1周内血糖急剧上升,波动幅度大,出现DM临床典型的三多一少症状,空腹血糖≥7.8 mmol/L,和（或）随机血糖≥17.8 mmol/L,2～3周左右逐步维持在稳定的中等程度高血糖状态.DM及DM＋CP组糖化血红蛋白水平[分别为（7.32±0.45）％、（9.41±0.45）％]均显著高于NC组[（4.02±0.45） ％]（P＜0.01）.复合模型大鼠（DM＋CP组）牙槽骨吸收最严重,第一、二磨牙牙齿间隙增宽,根分叉暴露.颈动脉病理结果显示DM＋CP组血管壁病变明显较其他组严重,血管壁变薄,弹性纤维断裂甚至部分消失,钙化严重,呈现典型的血管硬化表现.实时荧光定量PCR结果显示4组均检测到Pg DNA片段,NC组和DM组检出均为3/7,CP组为6/7,DM＋CP组为7/7.定量分析结果显示,CP组及DM＋CP组细菌含量均显著高于NC组和DM组（P＜0.05,P＜0.01）.结论 本实验成功建立2型DM＋CP大鼠模型,该复合模型颈动脉血管均出现钙化、纤维紊乱等典型的血管硬化病变,且颈动脉分叉处血管均检出Pg,含量也最高.Pg可能参与了DM血管的病变过程.

Objectives To establish SD rat model with type 2 diabetes mellitus （DM） and concomitant chronic periodontitis （CP） and to evaluate the influence of periodontitis on the vascular lesions of type 2 diabetes rats.Methods Totally 241 clean level SD rats were randomly divided into four groups,group A （normal control,NC,n=27）,group B （DM,n=34）,group C （CP,n=90） and group D （DM＋CP,n=90）.The rats of DM group were fed with high-fat and high-sugar diet for 8 to 10 weeks,and then were multiply injected with small dose streptozotocin under the condition of ice bath.Blood sugar levels after the injection were dynamically monitored at 72 h,1 week,2 weeks and 4 weeks,respectively.The CP model was established by means of ligation.Bilateral maxillary first and second molars were selected and ligated using 0.2 mm orthodontic wires binding with 4-0 surgical suture soaked with Porphyromonas gingivalis （Pg） suspension.After a period of 14 weeks,all the rats were put to death.Maxillary samples were subjected to methylene blue staining to observe alveolar bone loss.Bilateral carotid artery specimens were collected.The left carotid artery specimens were used to detect the prevalence of Pg using quantitative real-time PCR.The right carotid artery specimens were used to observe pathological changes.Results Blood sugar levels of rats in group B and D increased and changed sharply after Streptozotocin injection with in 1 week.Symptoms of ‘more drink,more food and body weight loss’ appeared.The fasting blood glucose （FBG） was more than 7.8 mmol/L and （or） the random blood glucose （RBG） was more than 17.8 mmol/L.Both FBG and RBG became stable after 2 to 3 weeks.Levels of HbA1C in group B and D （[7.32±0.45]%,[9.41±0.45]%） were significantly higher than that of group A （[4.02±0.45]%） （P〈0.01）.Rats of group D were observed the most severe bone loss showing wider interdental space and furcation involvement.Pathological results of carotid artery tissues of group D showed the worst lesions including thinning and calcification of vessel walls,and breaking down or disappearance of elastic fibers.The prevalences of DNA of Pg in groups of A,B,C and D were 3/7,3/7,6/7 and 7/7,respectively.The bacteria numbers detected by quantitative real-time PCR in groups C and D were significantly higher than that of groups A and B （P〈0.01）.Conclusions Rat model of type 2 DM with periodontitis was successfully established in the present study.Carotid artery specimens from DM ＋ CP model rats showed typical vascular lesions such as calcification and fiber disorders.Pg was found in all carotid specimens and the highest bacteria numbers were detected in the composite model rats.The Pg might play a role in the progress of diabetes vascular lesions.