牙龈卟啉单胞菌菌毛蛋白诱导内皮细胞炎症反应的信号通路研究
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摘要
目的：比较分析不同fimA型P. gingivalis菌毛蛋白诱导人脐静脉血管内皮细胞(human umbilical vein endothelial cells, HUVECs)炎症反应的相关信号转导。
方法：1.厌氧培养并鉴定ⅡfimA型(WCSP 115)和IV fimA型(W83)p.gingivalis,收集菌液提取细菌DNA,克隆ⅡfimA型和ⅣfimA型P.gingivalis的fimA序列,构建fimA的原核表达系统pET-30a-FimA BL21DE3, IPTG诱导表达重组菌毛蛋白rFimA;2.体外培养HUVECs(CRL-2480),待HUVECs单层融合后,与不同终浓度的P. gingivalis-rFimA(0.5μg/ml、5μg/ml、10μg/ml)共同孵育2h、6h、24h,提取HUVECs总RNA, Real Time-PCR检测CD14、TLR2(Toll-like receptor 2)、TLR4(Toll-like receptor 4)和MyD88(myeloid differentiation factor 88)的mRNA表达水平,流式细胞术(FCM)检测HUVECs膜表面CD14、跨膜受体TLR2和TLR4蛋白表达量。
结果：1.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs, CD14 mRNA表达量增加,且在2h时,Ⅳ型rFimA蛋白诱导HUVECs表达CD 14 mRNA的水平高于Ⅱ型rFimA蛋白(P<0.05)；24h时,Ⅳ型rFimA蛋白在三种浓度下诱导的CD14蛋白水平较阴性对照组的CD14蛋白水平升高(P<0.05),但2h时,5μg/ml与10μg/ml的Ⅳ型rFimA蛋白诱导的CD14蛋白水平降低,与基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与在6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。2.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR2 mRNA水平升高(P<0.05),且Ⅳ型rFimA诱导HUVECs的TLR2 mRNA表达水平高于Ⅱ型rFimA; 24h时,Ⅳ型rFimA的刺激使TLR2 mRNA水平升高并具有浓度依赖性,且各浓度组诱导的TLR2蛋白水平升高(P<0.05),与TLR2基因水平相符；但2h时,Ⅳ型rFimA 5μg/ml与10μg/ml浓度组诱导的TLR2蛋白水平均降低,与其基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的TLR2蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。3.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR4 mRNA水平升高(P<0.05),且Ⅳ型rFimA的三个浓度组相比较,5μg/ml组的TLR4 mRNA水平在三个时间点都达到峰值,但各浓度组的TLR4蛋白水平在各时间点均未见表达(P>0.05)。4.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs后,MyD88 mRNA表达量增加(P<0.05),且Ⅱ型P. gingivalis-rFimA的刺激存在着时间依赖效应。
结论：ⅡfimA型和ⅣfimA型P. gingivalis-FimA均能刺激HUVECs表达膜分子CD14和跨膜分子TLR2,而且使胞内分子MyD88和跨膜分子TLR4的mRNA转录水平升高,提示P. gingivalis可通过其菌毛蛋白FimA诱导内皮细胞的炎症反应,P.gingivalis-FimA可能是动脉粥样硬化的危险因素,并且可能是牙周炎与As相关的生物学基础；CD14-TLR2系统及MyD88信号通路可能参与了P. gingivalis菌毛诱导的内皮细胞炎症反应和细胞因子产生；不同fimA基因型P. gingivalis-rFimA对HUVECs功能的影响存在着差异,且Ⅳ型P. gingivalis-FimA诱发HUVECs炎症反应的能力强于Ⅱ型P. gingivalis-FimA。
Objective:To explore the intracellular signaling pathways involved in inflammatory response in HUVECs under the induction of fimbrillin from different strains of P. gingivalis with different fimA genotypes.
Methods:1. P. gingivalis WCSP 115 (typeⅡfimA gene) and W83 (typeⅣfimA gene)were cultured anaerobically in standard condition and identified,then P. gingivalis cells were collected to isolate their DNA.After the fimA gene sequence was cloned, we established fimA prokaryotic expression system pET-30a-FimA BL21DE3 which expressing recombinant fimbrillin under the induction of IPTG..2. HUVECs were cultured in vitro and different concentrations of recombinant fimbrillin from P. gingivalis with different fimA genotypes were added to confluent HUVECs monolayers and co-cultured for 2h,6h and 24h. At different time periods, HUVECs were collected, total RNA was extracted from part of them and used for RT-PCR to investigate the levels of mRNA of cluster of differentiation 14(CD14), Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4) and myeloid differentiation factor 88(MyD88).The other cells were detected by FCM analysis for CD14,TLR2,TLR4 expression.
Results:1. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of CD 14 mRNA expressed by HUVECs were higher than that of the negative control group(P<0.05). At 2h,the up-regulating effects on CD 14 mRNA expression by P. gingivalis-rFimA withⅣfimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype. After HUVECs were exposed to different concentrations(0.5μg/ml,5μg/ml,10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype for 24h, the production of CD 14 protein by HUVECs was higher than that of the negative control group(P<0.05). At 2h, however, when the higher dose (5μg/ml or lOμg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used, the level of CD14 protein producted by HUVECs was lower, and there was a discrepancy between the level of CD14 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h, and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of CD14 were similar to that of the negative control group (P>0.05).2. The expression of TLR2 mRNA in HUVECs induced by P. gingivalis-rFimA withⅡorⅣfimA genotype were higher than that of the negative control (p<0.05). The up-regulating effects on TLR2 mRNA by P. gingivalis-rFimA with IV fimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype.The up-regulating effects of TLR2 mRNA induced by P. gingivalis-rFimA with IV fimA genotype were in a dose-dependent manner at 24h. In addition, the level of TLR2 protein expressed by HUVECs stimulated with rFimA was higher than that of the negative control group(P<0.05),which was correspond to the gene level; At 2h, however, when the higher dose (5μg/ml or 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used,the level of TLR2 protein was lower, and there was a discrepancy between the level of TLR2 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h,and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of TLR2 were similar to that of the negative control group(P>0.05).3. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of TLR4 mRNA expressed by HUVECs was higher than that of the negative control(P<0.05). As compared with the stimulation of other concentrations (0.5μg/ml and 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype, the level of TLR4 mRNA reached its maximum with the stimulation of 5μg/ml P. gingivalis-rFimA withⅣfimA genotype at each time point. The level of TLR4 protein expressed by HUVECs were analyzed at 2h,6h and 24h, we observed that P. gingivalis-rFimA failed to stimulate TLR4 expression in HUVECs in each concentration group(p>0.05).4. When HUVECs were treated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of MyD88 mRNA expressed by HUVECs was higher than that of the negative control group(P<0.05). The up-regulating effects on MyD88 mRNA by P. gingivalis withⅡfimA genotype were in a time-dependent manner.
Conclusion:Based on these findings, we concluded that P. gingivalis-FimA withⅡorⅣfimA genotype could active HUVECs via expressing CD14 and TLR2 and up-regulating the level of MyD88 mRNA and TLR4 mRNA. It indicated that the inflammation response in HUVECs induced by P. gingivalis may be mediated through its FimA,A,P. gingivalis-FimA is likely a potential risk factor for atherosclerosis and the biological basis underlying the relationship between periodontitis and atherosclerosis.Our data suggest that singnaling pathways of CD14-TLR2 system and MyD88 may be involved in inflammatory response and cytokine production in HUVECs.The difference of P. gingivalis-FimA with fimA genotypes may lead different effects to HUVECs, and the stimulation of P. gingivalis-FimA withⅣfimA genotype was stronger than that of p. gingivalis-FimA withⅡfimA genotype.
方法：1.厌氧培养并鉴定ⅡfimA型(WCSP 115)和IV fimA型(W83)p.gingivalis,收集菌液提取细菌DNA,克隆ⅡfimA型和ⅣfimA型P.gingivalis的fimA序列,构建fimA的原核表达系统pET-30a-FimA BL21DE3, IPTG诱导表达重组菌毛蛋白rFimA;2.体外培养HUVECs(CRL-2480),待HUVECs单层融合后,与不同终浓度的P. gingivalis-rFimA(0.5μg/ml、5μg/ml、10μg/ml)共同孵育2h、6h、24h,提取HUVECs总RNA, Real Time-PCR检测CD14、TLR2(Toll-like receptor 2)、TLR4(Toll-like receptor 4)和MyD88(myeloid differentiation factor 88)的mRNA表达水平,流式细胞术(FCM)检测HUVECs膜表面CD14、跨膜受体TLR2和TLR4蛋白表达量。
结果：1.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs, CD14 mRNA表达量增加,且在2h时,Ⅳ型rFimA蛋白诱导HUVECs表达CD 14 mRNA的水平高于Ⅱ型rFimA蛋白(P<0.05)；24h时,Ⅳ型rFimA蛋白在三种浓度下诱导的CD14蛋白水平较阴性对照组的CD14蛋白水平升高(P<0.05),但2h时,5μg/ml与10μg/ml的Ⅳ型rFimA蛋白诱导的CD14蛋白水平降低,与基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与在6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。2.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR2 mRNA水平升高(P<0.05),且Ⅳ型rFimA诱导HUVECs的TLR2 mRNA表达水平高于Ⅱ型rFimA; 24h时,Ⅳ型rFimA的刺激使TLR2 mRNA水平升高并具有浓度依赖性,且各浓度组诱导的TLR2蛋白水平升高(P<0.05),与TLR2基因水平相符；但2h时,Ⅳ型rFimA 5μg/ml与10μg/ml浓度组诱导的TLR2蛋白水平均降低,与其基因表达水平不一致,Ⅳ型rFimA在2h的0.5μg/ml浓度组与6h时的三个浓度组、Ⅱ型rFimA各浓度组在三个时间点的TLR2蛋白水平与阴性对照组比较差异均无统计学意义(P>0.05)。3.Ⅱ型或Ⅳ型rFimA均能诱导HUVECs表达TLR4 mRNA水平升高(P<0.05),且Ⅳ型rFimA的三个浓度组相比较,5μg/ml组的TLR4 mRNA水平在三个时间点都达到峰值,但各浓度组的TLR4蛋白水平在各时间点均未见表达(P>0.05)。4.Ⅱ型或Ⅳ型rFimA蛋白刺激HUVECs后,MyD88 mRNA表达量增加(P<0.05),且Ⅱ型P. gingivalis-rFimA的刺激存在着时间依赖效应。
结论：ⅡfimA型和ⅣfimA型P. gingivalis-FimA均能刺激HUVECs表达膜分子CD14和跨膜分子TLR2,而且使胞内分子MyD88和跨膜分子TLR4的mRNA转录水平升高,提示P. gingivalis可通过其菌毛蛋白FimA诱导内皮细胞的炎症反应,P.gingivalis-FimA可能是动脉粥样硬化的危险因素,并且可能是牙周炎与As相关的生物学基础；CD14-TLR2系统及MyD88信号通路可能参与了P. gingivalis菌毛诱导的内皮细胞炎症反应和细胞因子产生；不同fimA基因型P. gingivalis-rFimA对HUVECs功能的影响存在着差异,且Ⅳ型P. gingivalis-FimA诱发HUVECs炎症反应的能力强于Ⅱ型P. gingivalis-FimA。
Objective:To explore the intracellular signaling pathways involved in inflammatory response in HUVECs under the induction of fimbrillin from different strains of P. gingivalis with different fimA genotypes.
Methods:1. P. gingivalis WCSP 115 (typeⅡfimA gene) and W83 (typeⅣfimA gene)were cultured anaerobically in standard condition and identified,then P. gingivalis cells were collected to isolate their DNA.After the fimA gene sequence was cloned, we established fimA prokaryotic expression system pET-30a-FimA BL21DE3 which expressing recombinant fimbrillin under the induction of IPTG..2. HUVECs were cultured in vitro and different concentrations of recombinant fimbrillin from P. gingivalis with different fimA genotypes were added to confluent HUVECs monolayers and co-cultured for 2h,6h and 24h. At different time periods, HUVECs were collected, total RNA was extracted from part of them and used for RT-PCR to investigate the levels of mRNA of cluster of differentiation 14(CD14), Toll-like receptor 2(TLR2), Toll-like receptor 4(TLR4) and myeloid differentiation factor 88(MyD88).The other cells were detected by FCM analysis for CD14,TLR2,TLR4 expression.
Results:1. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of CD 14 mRNA expressed by HUVECs were higher than that of the negative control group(P<0.05). At 2h,the up-regulating effects on CD 14 mRNA expression by P. gingivalis-rFimA withⅣfimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype. After HUVECs were exposed to different concentrations(0.5μg/ml,5μg/ml,10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype for 24h, the production of CD 14 protein by HUVECs was higher than that of the negative control group(P<0.05). At 2h, however, when the higher dose (5μg/ml or lOμg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used, the level of CD14 protein producted by HUVECs was lower, and there was a discrepancy between the level of CD14 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h, and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of CD14 were similar to that of the negative control group (P>0.05).2. The expression of TLR2 mRNA in HUVECs induced by P. gingivalis-rFimA withⅡorⅣfimA genotype were higher than that of the negative control (p<0.05). The up-regulating effects on TLR2 mRNA by P. gingivalis-rFimA with IV fimA genotype were stronger than that by P. gingivalis-rFimA withⅡfimA genotype.The up-regulating effects of TLR2 mRNA induced by P. gingivalis-rFimA with IV fimA genotype were in a dose-dependent manner at 24h. In addition, the level of TLR2 protein expressed by HUVECs stimulated with rFimA was higher than that of the negative control group(P<0.05),which was correspond to the gene level; At 2h, however, when the higher dose (5μg/ml or 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype was used,the level of TLR2 protein was lower, and there was a discrepancy between the level of TLR2 protein and the level of its mRNA. When HUVECs were stimulated with 0.5μg/ml rFimA from P. gingivalis withⅣfimA genotype at 2h,0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅣfimA genotype at 6h,and 0.5μg/ml,5μg/ml or 10μg/ml rFimA from P. gingivalis withⅡfimA genotype at 2h,6h and 24h, the protein expression of TLR2 were similar to that of the negative control group(P>0.05).3. When HUVECs were stimulated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of TLR4 mRNA expressed by HUVECs was higher than that of the negative control(P<0.05). As compared with the stimulation of other concentrations (0.5μg/ml and 10μg/ml) of P. gingivalis-rFimA withⅣfimA genotype, the level of TLR4 mRNA reached its maximum with the stimulation of 5μg/ml P. gingivalis-rFimA withⅣfimA genotype at each time point. The level of TLR4 protein expressed by HUVECs were analyzed at 2h,6h and 24h, we observed that P. gingivalis-rFimA failed to stimulate TLR4 expression in HUVECs in each concentration group(p>0.05).4. When HUVECs were treated with the P. gingivalis-rFimA withⅡorⅣfimA genotype, the level of MyD88 mRNA expressed by HUVECs was higher than that of the negative control group(P<0.05). The up-regulating effects on MyD88 mRNA by P. gingivalis withⅡfimA genotype were in a time-dependent manner.
Conclusion:Based on these findings, we concluded that P. gingivalis-FimA withⅡorⅣfimA genotype could active HUVECs via expressing CD14 and TLR2 and up-regulating the level of MyD88 mRNA and TLR4 mRNA. It indicated that the inflammation response in HUVECs induced by P. gingivalis may be mediated through its FimA,A,P. gingivalis-FimA is likely a potential risk factor for atherosclerosis and the biological basis underlying the relationship between periodontitis and atherosclerosis.Our data suggest that singnaling pathways of CD14-TLR2 system and MyD88 may be involved in inflammatory response and cytokine production in HUVECs.The difference of P. gingivalis-FimA with fimA genotypes may lead different effects to HUVECs, and the stimulation of P. gingivalis-FimA withⅣfimA genotype was stronger than that of p. gingivalis-FimA withⅡfimA genotype.
引文
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24.肖莉.ⅣfimA型牙龈卟啉单胞菌重组菌毛蛋白rFimA对血管内皮细胞功能影响的研究：[硕士学位论文].贵州：遵义医学院,2009.
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26. Zhou Q, Desta T, Fenton M,et al. Cytokine profiling of macrophages exposed to Porphyromonas gingivalis, its lipopolysaccharide, or its FimA protein.Infect Immun, 2005,73 (2):935-943.
27. Rudolph D, Yeh WC,Wakeham A, et al. Severe liver degeneration and lack of NF-kappaB activation in NEMO/IKK gamma-deficient mice[J]. Genes Dev,2000,14: 854-852.
28. Mehmet A. Eskan, George Hajishengallis, and Denis F. Kinane.Differential Activation of Human Gingival Epithelial Cells and Monocytes by Porphyromonas gingivalis Fimbriae. Infect Immun.2007,75(2):892-898.
29. Zhou Q, Amar S. Identification of signaling pathways in macrophage exposed to Porphyromonas gingivalis or to its purified cell wall components. The Journal of Immunology,2007,179,7777-7790
30. Frank C.Gibson,et a .Innate immune recognition of invasive bacterial accelerates atherosclerosis in apolipoprotein E-deficient mice. Circulation.2004 Jun 8,109(22):2801-6.
31. Shapira L, Takashiba S, Amar S, Van Dyke T. Porphyromonas gingivalis lipopolysaccharide stimulation of human monocytes:dependence on serum and CD14 receptor. Oral Microbiol Immunol 994,9:112-117.
32. Ogawa T, Uchida H, Hamada S. Porphyromonas gingivalis fimbriae and their synthetic peptides induce proinflammatory cytokines in human peripheral blood monocyte cultures. FEMS Microbiol Lett 1994,116:237-242.
33. Amano A, Sharma A, Lee J-Y, Sojar H, Raj P, Genco R. Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin. Infect Immun 1996,64:1631-1637.
34. Njoroge T, Genco R, Sojar H, Hamada N, Genco C. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.Infect Immun 1997,65: 1980-1984.
35. Malek R, Fisher G, Caleca A et al. Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. J Bacteriol 1994,176: 1052-1059.
36. Hashimoto M, Ogawa S, Asai Y, Takai Y, Ogawa T. Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin. FEMS Microbiol Lett 2003,226: 267-271.
37. Hamada N, Watanabe K, Sasakawa C, Yoshikawa M, Yoshimura F, Umemoto T. Construction and characterization of a fimAmutant of Porphyromonas gingivalis. Infect Immun 1994,62:1696-1704.
38. Weinberg A, Belton C, Park Y, Lamont R. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 1997,65:313-316.
39. Dana T. Graves,et al.Porphyromonas gingivalis fimbriae are pro-inflammatory but do not play a prominent role in the innate immune response to P. gingivalis. Journal of Endotoxin Research 2005 11:13.
40. Haraszthy VI, Zambon JJ, Trevisan M et al. Identification of periodontal pathogens in atheromatous plaques. J, Periodontol.2000 Oct; 71 (10):1554-1560.
41. Chiu B. Multiple infections in carotid atherosclerotic plaques Am Heart J.1999 Nov; 138(5 pt 2):s534-536.
42. Khlgatian M, Nassar H, Chou HH et al. Fimbrae-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells. Infect Immun.2002 Jan; 70(1):257-267
(1)邹智.内皮素1与牙周炎.国外医学口腔医学分册,2003,33(2)
(2)Ugar-Cankal D, Ozmeric N. A multifaceted molecule, nitric oxide in oral and periodontal diseases. Clin Chim Acta.2006 Apr; 366(1-2):90-100. Epub 2006 Jan 4.
(3)Kim SJ, Choi EY, Cho YJ, et al. Surface-associated material from Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthase. Microbes Infect.2006,8(2):470-7. Epub 2005 Sep 19
(4)Yamampto E,AwanoS, et al.J Peridontal Res,2003,38(4):417-421
(5)Despade RG,Khan MB,Genco CA.Invision of aortic and heart endothelial cells by Porphyromonas gingivalis Infect Immune,1998,66,(11):5337-3543
(6)吴娟,孙卫斌,季勇.牙龈卟啉单胞菌对人脐静脉内皮细胞一氧化氮生成的影响.中华口腔医学杂志,2009,44(1)
(7)俞梦越,吴力.心功能不全与冠心病、心血管病学进展[J].2002,4(23)：193-199
(8)Wang JJ,Liu SM,Chen R,et al.A novel effect lobeline on vascular smooth muscle cell:inhibition of proliferation induced by endothelin21 [J] Pharmazie,2007,62, (8): 620-624
(9)Fakhouri F,Placiers,Ardaillou R.et al Angiotensin Ⅱacti-vates colleagen type I gene in the renal cortex and aorta of transgenic mice through interaction with endothelin and TGF-β JAM Soc Nephrol,2001,12:2701
(10)徐正平,内皮素转化酶-内皮素与动脉粥样硬化关系的研究进展,南通大学学报,2005,25(2)
(11)Howard K,Role for Periodontal Bacterial in Cardiovascular Disease,2001;6:41-47
(12)Gibson FC, Yumoto H, Takahashi Y, et al. Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis. J Dent Res2006,85(2):106-121
(13)Takahashi Y, Davey M, Yumoto H, et al. Fimbriae-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells. Cell Microbiol.2006,8(5):738-757
(14)Southerland JH, Taylor GW, Moss K, et al. Commonality in chronic inflammatory diseases:periodontitis, diabetes, and coronary artery disease. Periodontol 2000. 2006,40:130-143
(15)Choi EY, Hwang YM, Lee JY, et al. Lipid A-associated proteins from Porphyromonas gingivalis stimulate release of nitric oxide by inducing expression of inducible nitric oxide synthase. J Periodontal Res.2007Aug; 42(4):350-60.
(16)Kendall HK, Haase HR, Li H, et al. Nitric oxide synthase type-Ⅱ is synthesized by human gingival tissue and cultured human gingival fibroblasts. J Periodontal Res.2000 Aug; 35(4):194-200.
(17)Batista AC, Silva TA, Chun JH, et al. Nitric oxide synthesis and severity of human periodontal disease. Oral Dis.2002 Sep; 8(5):254-60.
(18)Beck JD, Offenbacher S. The association between periodontal diseases and cardiovascular diseases:a state-of-the-science review. Ann Periodontol.2001; 6(1):9-15
(19)Scannapieco FA, Bush RB, Paju S. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke. A systemic review. Ann Periodontol.2003,8:38-53
(20)Libby P. Inflammation in atherosclerosis. Nature 2002;420:868-874
(21)Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med 1999,340(2): 115-126
(22)Friedewald VE, Kornman KS, Beck JD, et al. The American Journal of Cardiology and Journal of Periodontology editor's consensus:periodontitis and atherosclerotic cardiovascular disease. J Periodontol.2009,80(7):1021-1032
(23)Zhou Q,et al.Singaling mechanisms involved in altered function of macrophages fron diet-induced obese mice affect immune response.Proc Natl Acad Sci USA.2009 Jun 30,106(26):10740-5
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8. Harokopakis E, Albzreh M, Martin M, et al. TLR2 Transmodulates Monocyte Adhesion and Transmigration via Racl-and P13K-Mediated Inside-Out Signaling in Response to Porphyromonas gingivalis Fimbriae.The Journal of Immunology,2006; 176 7645-7656.
9. Triantafilou, Gamper F, Lepper P, et al. Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in humanvascular endothelial cells.Cellular Microbiology 2007,9(8),2030-2039
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11.郭永华,吴亚菲,刘天佳等.不同fimA基因型牙龈卟啉单胞菌在慢性牙周炎患者中的分布[J].华西口腔医学杂志,2005,23(2)：99-102
12. Ferdaus Hassan, Shamima Islam, Gantsetseg Tumurkhuu, Yoshikazu Naiki, et al. Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide. BMC Cancer 2006,6:281
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17. Lien, E., T. J. Sellati, A. Yoshimura, T. H. Flo, G. Rawadi, R. W. Finberg, J. D. Carroll, T. Espevik, R. R. Ingalls, J. D. Radolf, and D. T. Golenbock.1999. Toll-like receptor 2 functions as a pattern recognition receptor for diverse bacterial products. J. Biol. Chem.274:33419-33425.
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19. Schwandner, R., R. Dziarski, H. Wesche, M. Rothe, and C. J. Kirschning.1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J. Biol. Chem.274:17406-17409.
20. George Hajishengallis,Michael Martin, Dependence of Bacterial Protein Adhesins on Toll-Like Receptors for Proinflammatory Cytokine Induction. Clin Diagn Lab Immunol.2002,9(2):403-411.
21. Ogawa, T., H. Uchida, and K. Amino.1994. Immunobiological activities of chemically defined lipid A from lipopolysaccharides of Porphyromonas gingivalis. Microbiology 140:1209-1216.
22. VirjiM,Makepeace K, Peak I R, et al. Opc-and p ilus-dependent interactions of meningococci with human epithelial cells:molecularmechanis ms and modulation by surface polysac2 charides[J]. MolM icrobiol,1995,18 (4):741-754
23. BRIGHT BILLHD, LIBRATY D H, KRUTZIK S R, et al.Host defense mechanisms triggered by microbial lipoproteins through Toll-Like receptors [J]1 Science,1999, 285:7322 7361
24.肖莉.ⅣfimA型牙龈卟啉单胞菌重组菌毛蛋白rFimA对血管内皮细胞功能影响的研究：[硕士学位论文].贵州：遵义医学院,2009.
25. Schilling DK, Thomas K, Nixdorff SN et al. Toll-like receptor 4 and Toll-IL-1 receptor domain-containing adapter protein (TIRAP)/myeloid differentiation protein 88 adapter-like (Mal) contribute to maximal IL-6 expression in macrophages. J. Immunol. 2002.169:5874-5880.
26. Zhou Q, Desta T, Fenton M,et al. Cytokine profiling of macrophages exposed to Porphyromonas gingivalis, its lipopolysaccharide, or its FimA protein.Infect Immun, 2005,73 (2):935-943.
27. Rudolph D, Yeh WC,Wakeham A, et al. Severe liver degeneration and lack of NF-kappaB activation in NEMO/IKK gamma-deficient mice[J]. Genes Dev,2000,14: 854-852.
28. Mehmet A. Eskan, George Hajishengallis, and Denis F. Kinane.Differential Activation of Human Gingival Epithelial Cells and Monocytes by Porphyromonas gingivalis Fimbriae. Infect Immun.2007,75(2):892-898.
29. Zhou Q, Amar S. Identification of signaling pathways in macrophage exposed to Porphyromonas gingivalis or to its purified cell wall components. The Journal of Immunology,2007,179,7777-7790
30. Frank C.Gibson,et a .Innate immune recognition of invasive bacterial accelerates atherosclerosis in apolipoprotein E-deficient mice. Circulation.2004 Jun 8,109(22):2801-6.
31. Shapira L, Takashiba S, Amar S, Van Dyke T. Porphyromonas gingivalis lipopolysaccharide stimulation of human monocytes:dependence on serum and CD14 receptor. Oral Microbiol Immunol 994,9:112-117.
32. Ogawa T, Uchida H, Hamada S. Porphyromonas gingivalis fimbriae and their synthetic peptides induce proinflammatory cytokines in human peripheral blood monocyte cultures. FEMS Microbiol Lett 1994,116:237-242.
33. Amano A, Sharma A, Lee J-Y, Sojar H, Raj P, Genco R. Structural domains of Porphyromonas gingivalis recombinant fimbrillin that mediate binding to salivary proline-rich protein and statherin. Infect Immun 1996,64:1631-1637.
34. Njoroge T, Genco R, Sojar H, Hamada N, Genco C. A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells.Infect Immun 1997,65: 1980-1984.
35. Malek R, Fisher G, Caleca A et al. Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. J Bacteriol 1994,176: 1052-1059.
36. Hashimoto M, Ogawa S, Asai Y, Takai Y, Ogawa T. Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin. FEMS Microbiol Lett 2003,226: 267-271.
37. Hamada N, Watanabe K, Sasakawa C, Yoshikawa M, Yoshimura F, Umemoto T. Construction and characterization of a fimAmutant of Porphyromonas gingivalis. Infect Immun 1994,62:1696-1704.
38. Weinberg A, Belton C, Park Y, Lamont R. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infect Immun 1997,65:313-316.
39. Dana T. Graves,et al.Porphyromonas gingivalis fimbriae are pro-inflammatory but do not play a prominent role in the innate immune response to P. gingivalis. Journal of Endotoxin Research 2005 11:13.
40. Haraszthy VI, Zambon JJ, Trevisan M et al. Identification of periodontal pathogens in atheromatous plaques. J, Periodontol.2000 Oct; 71 (10):1554-1560.
41. Chiu B. Multiple infections in carotid atherosclerotic plaques Am Heart J.1999 Nov; 138(5 pt 2):s534-536.
42. Khlgatian M, Nassar H, Chou HH et al. Fimbrae-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells. Infect Immun.2002 Jan; 70(1):257-267
(1)邹智.内皮素1与牙周炎.国外医学口腔医学分册,2003,33(2)
(2)Ugar-Cankal D, Ozmeric N. A multifaceted molecule, nitric oxide in oral and periodontal diseases. Clin Chim Acta.2006 Apr; 366(1-2):90-100. Epub 2006 Jan 4.
(3)Kim SJ, Choi EY, Cho YJ, et al. Surface-associated material from Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthase. Microbes Infect.2006,8(2):470-7. Epub 2005 Sep 19
(4)Yamampto E,AwanoS, et al.J Peridontal Res,2003,38(4):417-421
(5)Despade RG,Khan MB,Genco CA.Invision of aortic and heart endothelial cells by Porphyromonas gingivalis Infect Immune,1998,66,(11):5337-3543
(6)吴娟,孙卫斌,季勇.牙龈卟啉单胞菌对人脐静脉内皮细胞一氧化氮生成的影响.中华口腔医学杂志,2009,44(1)
(7)俞梦越,吴力.心功能不全与冠心病、心血管病学进展[J].2002,4(23)：193-199
(8)Wang JJ,Liu SM,Chen R,et al.A novel effect lobeline on vascular smooth muscle cell:inhibition of proliferation induced by endothelin21 [J] Pharmazie,2007,62, (8): 620-624
(9)Fakhouri F,Placiers,Ardaillou R.et al Angiotensin Ⅱacti-vates colleagen type I gene in the renal cortex and aorta of transgenic mice through interaction with endothelin and TGF-β JAM Soc Nephrol,2001,12:2701
(10)徐正平,内皮素转化酶-内皮素与动脉粥样硬化关系的研究进展,南通大学学报,2005,25(2)
(11)Howard K,Role for Periodontal Bacterial in Cardiovascular Disease,2001;6:41-47
(12)Gibson FC, Yumoto H, Takahashi Y, et al. Innate immune signaling and Porphyromonas gingivalis-accelerated atherosclerosis. J Dent Res2006,85(2):106-121
(13)Takahashi Y, Davey M, Yumoto H, et al. Fimbriae-dependent activation of pro-inflammatory molecules in Porphyromonas gingivalis infected human aortic endothelial cells. Cell Microbiol.2006,8(5):738-757
(14)Southerland JH, Taylor GW, Moss K, et al. Commonality in chronic inflammatory diseases:periodontitis, diabetes, and coronary artery disease. Periodontol 2000. 2006,40:130-143
(15)Choi EY, Hwang YM, Lee JY, et al. Lipid A-associated proteins from Porphyromonas gingivalis stimulate release of nitric oxide by inducing expression of inducible nitric oxide synthase. J Periodontal Res.2007Aug; 42(4):350-60.
(16)Kendall HK, Haase HR, Li H, et al. Nitric oxide synthase type-Ⅱ is synthesized by human gingival tissue and cultured human gingival fibroblasts. J Periodontal Res.2000 Aug; 35(4):194-200.
(17)Batista AC, Silva TA, Chun JH, et al. Nitric oxide synthesis and severity of human periodontal disease. Oral Dis.2002 Sep; 8(5):254-60.
(18)Beck JD, Offenbacher S. The association between periodontal diseases and cardiovascular diseases:a state-of-the-science review. Ann Periodontol.2001; 6(1):9-15
(19)Scannapieco FA, Bush RB, Paju S. Associations between periodontal disease and risk for atherosclerosis, cardiovascular disease, and stroke. A systemic review. Ann Periodontol.2003,8:38-53
(20)Libby P. Inflammation in atherosclerosis. Nature 2002;420:868-874
(21)Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med 1999,340(2): 115-126
(22)Friedewald VE, Kornman KS, Beck JD, et al. The American Journal of Cardiology and Journal of Periodontology editor's consensus:periodontitis and atherosclerotic cardiovascular disease. J Periodontol.2009,80(7):1021-1032
(23)Zhou Q,et al.Singaling mechanisms involved in altered function of macrophages fron diet-induced obese mice affect immune response.Proc Natl Acad Sci USA.2009 Jun 30,106(26):10740-5