【摘要】：目的:分析經(jīng)牙周感染后牙齦卟啉單胞菌(Porphyromonas gingivalis,P.gingivali s)對(duì)載脂蛋白E基因敲除(apolipoprotein E gene knock out,Apo E-/-)小鼠主動(dòng)脈粥樣硬化斑塊的形成、以及對(duì)血管內(nèi)皮細(xì)胞(vascular endothelial cells,VECs)天然免疫信號(hào)轉(zhuǎn)導(dǎo)相關(guān)受體、核因子κB(nuclear factorκB,NF-κB)轉(zhuǎn)錄因子和對(duì)血管平滑肌細(xì)胞(vascular Smooth muscle cells,VSMCs)增殖與遷移的影響,探討在動(dòng)脈粥樣硬化(atherosclerosis,As)發(fā)生發(fā)展中P.gingivalis的可能作用和相關(guān)機(jī)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(duì)Apo E-/-小鼠上頜第二磨牙牙頸部進(jìn)行絲線結(jié)扎+涂菌法構(gòu)建牙周炎動(dòng)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(cè)上頜骨,一側(cè)用于制作牙周組織病理切片,通過HE染色對(duì)其進(jìn)行組織學(xué)檢查,另一側(cè)則在體式顯微鏡下觀察牙槽骨吸收的情況;并對(duì)小鼠主動(dòng)脈組織取材,通過16S r DNA聚合酶鏈反應(yīng)法檢測(cè)P.gingivalis在主動(dòng)脈中的含量,并進(jìn)行半定量分析;制作主動(dòng)脈組織冰凍切片,HE染色和油紅O染色觀察主動(dòng)脈斑塊形成情況,免疫組織化學(xué)染色法檢測(cè)VECs表面TLR2、TLR4的表達(dá)、NF-κB的核移位以及VSMCs的增殖和遷移情況。結(jié)果:(1)本實(shí)驗(yàn)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(shí)驗(yàn)。(2)實(shí)驗(yàn)組小鼠出現(xiàn)明顯牙周炎癥狀,上頜第二磨牙近遠(yuǎn)中鄰面軟組織退縮,牙槽嵴頂降低,牙槽骨吸收嚴(yán)重;牙周組織HE染色顯示,上皮表面糜爛,上皮釘突局部呈網(wǎng)狀增生,并伴新生毛細(xì)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(shí)驗(yàn)組上頜第二磨牙牙周組織破壞嚴(yán)重,牙槽骨吸收至根尖1/3,而對(duì)照組則無(wú)明顯異常改變;(3)在所有主動(dòng)脈標(biāo)本中,均可檢測(cè)到P.gingivalis的存在,經(jīng)統(tǒng)計(jì)學(xué)分析,實(shí)驗(yàn)組的P.gingivalis的相對(duì)含量為(1.274±0.637)、顯著高于對(duì)照組(0.115±0.025),且差異均具有統(tǒng)計(jì)學(xué)意義(P0.01);(4)主動(dòng)脈冰凍切片HE染色和油紅O染色結(jié)果表明,實(shí)驗(yàn)組小鼠主動(dòng)脈形成典型As斑塊,經(jīng)統(tǒng)計(jì)學(xué)分析,HE染色中實(shí)驗(yàn)組斑塊面積為(0.449±0.018)mm2、明顯高于對(duì)照組(0.338±0.021)mm2,油紅O染色實(shí)驗(yàn)組斑塊面積為(1.815±0.076)m m2、明顯高于對(duì)照組(1.339±0.082)mm2,且差異均具有統(tǒng)計(jì)學(xué)意義(P㩳0.05);(5)免疫組織化學(xué)染色顯示實(shí)驗(yàn)組主動(dòng)脈VSMC由中膜向內(nèi)膜遷移和增殖的強(qiáng)度顯著高于對(duì)照組;內(nèi)皮細(xì)胞上的TLR2、TLR4的強(qiáng)陽(yáng)性表達(dá)的范圍明顯高于對(duì)照組,且NF-κB核移位的表達(dá)強(qiáng)于對(duì)照組。結(jié)論:牙周感染的P.gingivalis可能播散至主動(dòng)脈血管壁組織,并通過影響主動(dòng)脈內(nèi)皮細(xì)胞Toll樣受體(Toll like receptors,TLRs)與NF-κB炎癥信號(hào)通路以及平滑肌細(xì)胞的增殖和遷移等,促進(jìn)Apo E-/-小鼠主動(dòng)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(xué)位授予單位】：遵義醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R781.4

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