【摘要】：目的:分析經牙周感染后牙齦卟啉單胞菌(Porphyromonas gingivalis,P.gingivali s)對載脂蛋白E基因敲除(apolipoprotein E gene knock out,Apo E-/-)小鼠主動脈粥樣硬化斑塊的形成、以及對血管內皮細胞(vascular endothelial cells,VECs)天然免疫信號轉導相關受體、核因子κB(nuclear factorκB,NF-κB)轉錄因子和對血管平滑肌細胞(vascular Smooth muscle cells,VSMCs)增殖與遷移的影響,探討在動脈粥樣硬化(atherosclerosis,As)發(fā)生發(fā)展中P.gingivalis的可能作用和相關機制,為As的早期防治提供新的思路。方法:厭氧培養(yǎng)P.gingivalis并通過革蘭染色予以鑒定;通過對Apo E-/-小鼠上頜第二磨牙牙頸部進行絲線結扎+涂菌法構建牙周炎動物模型,高脂飼料喂養(yǎng),12周后安樂處死小鼠。截取小鼠雙側上頜骨,一側用于制作牙周組織病理切片,通過HE染色對其進行組織學檢查,另一側則在體式顯微鏡下觀察牙槽骨吸收的情況;并對小鼠主動脈組織取材,通過16S r DNA聚合酶鏈反應法檢測P.gingivalis在主動脈中的含量,并進行半定量分析;制作主動脈組織冰凍切片,HE染色和油紅O染色觀察主動脈斑塊形成情況,免疫組織化學染色法檢測VECs表面TLR2、TLR4的表達、NF-κB的核移位以及VSMCs的增殖和遷移情況。結果:(1)本實驗所培養(yǎng)的P.gingivalis在BHI血瓊脂平板上呈現(xiàn)特征性的黑色圓形光滑菌落,油鏡下見P.gingivalis為G-球桿菌,為純培養(yǎng)物,可用于后續(xù)實驗。(2)實驗組小鼠出現(xiàn)明顯牙周炎癥狀,上頜第二磨牙近遠中鄰面軟組織退縮,牙槽嵴頂降低,牙槽骨吸收嚴重;牙周組織HE染色顯示,上皮表面糜爛,上皮釘突局部呈網狀增生,并伴新生毛細血管和膠原纖維增生。體視顯微鏡下觀察牙槽骨吸收程度,實驗組上頜第二磨牙牙周組織破壞嚴重,牙槽骨吸收至根尖1/3,而對照組則無明顯異常改變;(3)在所有主動脈標本中,均可檢測到P.gingivalis的存在,經統(tǒng)計學分析,實驗組的P.gingivalis的相對含量為(1.274±0.637)、顯著高于對照組(0.115±0.025),且差異均具有統(tǒng)計學意義(P0.01);(4)主動脈冰凍切片HE染色和油紅O染色結果表明,實驗組小鼠主動脈形成典型As斑塊,經統(tǒng)計學分析,HE染色中實驗組斑塊面積為(0.449±0.018)mm2、明顯高于對照組(0.338±0.021)mm2,油紅O染色實驗組斑塊面積為(1.815±0.076)m m2、明顯高于對照組(1.339±0.082)mm2,且差異均具有統(tǒng)計學意義(P㩳0.05);(5)免疫組織化學染色顯示實驗組主動脈VSMC由中膜向內膜遷移和增殖的強度顯著高于對照組;內皮細胞上的TLR2、TLR4的強陽性表達的范圍明顯高于對照組,且NF-κB核移位的表達強于對照組。結論:牙周感染的P.gingivalis可能播散至主動脈血管壁組織,并通過影響主動脈內皮細胞Toll樣受體(Toll like receptors,TLRs)與NF-κB炎癥信號通路以及平滑肌細胞的增殖和遷移等,促進Apo E-/-小鼠主動脈As的發(fā)生發(fā)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 鹵0.637), which was significantly higher than that in the control group (0.115 鹵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 鹵0.018) mm2, in HE staining. The plaque area of the experimental group was (1.815 鹵0.076) mm ~ 2 and (1.339 鹵0.082) mm2, significantly higher than that of the control group (0.338 鹵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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R781.4

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