P.gingivalis感染对小鼠动脉粥样硬化形成的研究

发布时间：2019-02-08 16:38

【摘要】：目的:分析经牙周感染后牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivali s)对载脂蛋白E基因敲除(apolipoprotein E gene knock out,Apo E-/-)小鼠主动脉粥样硬化斑块的形成、以及对血管内皮细胞(vascular endothelial cells,VECs)天然免疫信号转导相关受体、核因子κB(nuclear factorκB,NF-κB)转录因子和对血管平滑肌细胞(vascular Smooth muscle cells,VSMCs)增殖与迁移的影响,探讨在动脉粥样硬化(atherosclerosis,As)发生发展中P.gingivalis的可能作用和相关机制,为As的早期防治提供新的思路。方法:厌氧培养P.gingivalis并通过革兰染色予以鉴定;通过对Apo E-/-小鼠上颌第二磨牙牙颈部进行丝线结扎+涂菌法构建牙周炎动物模型,高脂饲料喂养,12周后安乐处死小鼠。截取小鼠双侧上颌骨,一侧用于制作牙周组织病理切片,通过HE染色对其进行组织学检查,另一侧则在体式显微镜下观察牙槽骨吸收的情况;并对小鼠主动脉组织取材,通过16S r DNA聚合酶链反应法检测P.gingivalis在主动脉中的含量,并进行半定量分析;制作主动脉组织冰冻切片,HE染色和油红O染色观察主动脉斑块形成情况,免疫组织化学染色法检测VECs表面TLR2、TLR4的表达、NF-κB的核移位以及VSMCs的增殖和迁移情况。结果:(1)本实验所培养的P.gingivalis在BHI血琼脂平板上呈现特征性的黑色圆形光滑菌落,油镜下见P.gingivalis为G-球杆菌,为纯培养物,可用于后续实验。(2)实验组小鼠出现明显牙周炎症状,上颌第二磨牙近远中邻面软组织退缩,牙槽嵴顶降低,牙槽骨吸收严重;牙周组织HE染色显示,上皮表面糜烂,上皮钉突局部呈网状增生,并伴新生毛细血管和胶原纤维增生。体视显微镜下观察牙槽骨吸收程度,实验组上颌第二磨牙牙周组织破坏严重,牙槽骨吸收至根尖1/3,而对照组则无明显异常改变;(3)在所有主动脉标本中,均可检测到P.gingivalis的存在,经统计学分析,实验组的P.gingivalis的相对含量为(1.274±0.637)、显著高于对照组(0.115±0.025),且差异均具有统计学意义(P0.01);(4)主动脉冰冻切片HE染色和油红O染色结果表明,实验组小鼠主动脉形成典型As斑块,经统计学分析,HE染色中实验组斑块面积为(0.449±0.018)mm2、明显高于对照组(0.338±0.021)mm2,油红O染色实验组斑块面积为(1.815±0.076)m m2、明显高于对照组(1.339±0.082)mm2,且差异均具有统计学意义(P㩳0.05);(5)免疫组织化学染色显示实验组主动脉VSMC由中膜向内膜迁移和增殖的强度显著高于对照组;内皮细胞上的TLR2、TLR4的强阳性表达的范围明显高于对照组,且NF-κB核移位的表达强于对照组。结论:牙周感染的P.gingivalis可能播散至主动脉血管壁组织,并通过影响主动脉内皮细胞Toll样受体(Toll like receptors,TLRs)与NF-κB炎症信号通路以及平滑肌细胞的增殖和迁移等,促进Apo E-/-小鼠主动脉As的发生发展。
[Abstract]:Objective: to study the formation of atherosclerotic plaque in aorta of apolipoprotein E knockout (apolipoprotein E gene knock out,Apo E-r- mice after periodontal infection with porphyromonas gingivalis (Porphyromonas gingivalis,P.gingivali s). And the effects on the innate immune signal transduction related receptors of vascular endothelial cells (vascular endothelial cells,VECs), nuclear factor 魏 B (NF- 魏 B) transcription factor and on the proliferation and migration of vascular smooth muscle cells (vascular Smooth muscle cells,VSMCs). To explore the possible role and related mechanism of P.gingivalis in the pathogenesis and development of atherosclerosis (atherosclerosis,As), and to provide new ideas for early prevention and treatment of As. Methods: P.gingivalis was cultured and identified by Gram staining. The animal model of periodontitis was established by filamentous ligation of the neck of the maxillary second molar of Apo E-r-mice. The animal model was fed with high fat diet, and the mice were killed 12 weeks later. The bilateral maxillary bones of mice were cut off and used to make pathological sections of periodontal tissues. The resorption of alveolar bone was observed by HE staining on the other side. The content of P.gingivalis in aorta was detected by 16s r DNA polymerase chain reaction and semi-quantitative analysis was carried out. The aortic plaques were observed by HE staining and oil red O staining. The expression of TLR2,TLR4 on the surface of VECs, the nuclear translocation of NF- 魏 B and the proliferation and migration of VSMCs were detected by immunohistochemical staining. Results: (1) the P.gingivalis cultured in this experiment showed a characteristic black round smooth colony on the BHI blood Agar plate. Under the oil microscope, P.gingivalis was found to be a G-spherobacterium and a pure culture. It can be used in the follow-up experiment. (2) the experimental group showed obvious periodontitis symptoms, the maxillary second molars receded in the proximal and middle sides, the alveolar crest decreased, and the alveolar bone resorption was serious. HE staining of periodontal tissue showed erosion of epithelial surface, local reticular hyperplasia of epithelial nailing, and proliferation of new capillaries and collagen fibers. The degree of alveolar bone resorption was observed under stereoscopic microscope. The periodontal tissue of the maxillary second molar in the experimental group was severely damaged, and the alveolar bone was absorbed to the apical root for one third of the time, while the control group had no obvious abnormal changes. (3) the presence of P.gingivalis was detected in all aortic specimens. The relative content of P.gingivalis in the experimental group was (1.274 卤0.637), which was significantly higher than that in the control group (0.115 卤0.025). The difference was statistically significant (P0.01). (4) the results of HE staining and oil red O staining on frozen sections of aorta showed that the typical As plaques were formed in the aorta of the experimental group. By statistical analysis, the plaque area of the experimental group was (0.449 卤0.018) mm2, in HE staining. The plaque area of the experimental group was (1.815 卤0.076) mm ~ 2 and (1.339 卤0.082) mm2, significantly higher than that of the control group (0.338 卤0.021) mm2, oil red O staining. (5) Immunohistochemical staining showed that the migration and proliferation of VSMC from medial membrane to intima in experimental group was significantly higher than that in control group. The range of strong TLR2,TLR4 positive expression in endothelial cells was significantly higher than that in the control group, and the expression of NF- 魏 B was stronger than that in the control group. Conclusion: periodontal infection of P.gingivalis may spread to aortic vascular wall, and influence the proliferation and migration of smooth muscle cells by affecting Toll like receptor (Toll like receptors,TLRs) and NF- 魏 B inflammatory signaling pathway in aortic endothelial cells. Promote the development of As in Apo E-r-mouse aorta.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R781.4

【相似文献】