牙龈卟啉单胞菌对人脐静脉血管内皮细胞cGMP生成的影响

目的:体外研究牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对人脐静脉血管内皮细胞(human umbilical vein endothelial cell,HUVEC)cGMP水平的影响。方法:用Pg ATCC 33277分别以感染复数(multiplicity of infection,MOI)1∶10、1∶50、1∶250干预HUVEC,并以未受Pg ATCC 33277干预的HUVEC作为阴性对照,分别培养4、12、36 h,在倒置显微镜下观察各组细胞形态;125I cGMP放射免疫试剂盒检测各组HUVECcGMP的水平。结果:与对照组相比,Pg ATCC 33277分别以MOI 1∶10、1∶50、1∶250干预HUVEC 4、12、36 h后,实验各组HUVEC的细胞形态未见明显改变,仍呈典型的"铺路石"状单层贴壁生长;Pg ATCC 33277 MOI1∶250时可呈时间依赖性地降低HUVEC cGMP水平,而同一时间点内,各浓度组的cGMP水平并无明显差异。结论:短时间内,Pg ATCC 33277对HUVEC的细胞形态无明显影响,但可降低HUVEC cGMP的生成,HUVECNO生物利用度下降。     

牙龈卟啉单胞菌入侵血管内皮细胞的实验研究

目的:体外观察牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖的影响,观察Pg对HUVEC的入侵能力,从而探讨Pg对内皮细胞功能损伤的途径,以期为牙周病在动脉粥样硬化(Atherosclerosis,As)发病机制中的作用提供依据。方法:用感染复数(multiplicity of infection,MOI)1:10,1:100PgATCC33277分别干预HUVEC 4h、8h、12h、24h,未受Pg ATCC33277干预的HUVEC作为阴性对照组,倒置显微镜下观察HUVEC形态,CCK-8法测定HUVEC细胞的增殖情况,透射电镜下观察Pg ATCC33277对HUVEC的入侵情况。结果:在24h内,与对照组相比,受Pg ATCC33277感染的HUVEC的细胞形态未见明显影响,仍呈典型的"铺路石"单层贴壁生长,Pg ATCC33277对HUVEC细胞增殖未见明显影响,透射电镜下观察发现Pg ATCC33277可以黏附HUVEC细胞膜表面,入侵HUVEC,直接定植或以空泡的形式存在于细胞的胞质中。结论:Pg可以入侵内皮细胞,以在细胞内定植的方式逃避宿主的防御反应,内皮细胞的形态和增殖未见明显改变,从而形成第二次慢性感染过程,进一步影响内皮细胞正常功能的发挥。     

牙龈卟啉单胞菌对兔血管平滑肌细胞分泌IL-1β、IL-6的影响

组织块法培养兔腹主动脉血管平滑肌细胞,并对其细胞来源进行鉴定。用4.3×106CFU/mL的Pg上清液刺激细胞12、24、48h后,通过ELISA检测IL-1β、IL-6的水平;同时用RT-PCR检测其mRNA表达的情况。结果:Pg上清液刺激24h和48h时能促进血管平滑肌细胞分泌IL-6,分别与同一时间点的对照组相比差异均有统计学意义(P<0.05),而且24h时,IL-6表达最强,而IL-1β的表达最低,明显低于相同时间的对照组和其他时间点的实验组(P<0.05)。RT-     

牙龈卟啉单胞菌对兔血管平滑肌细胞分泌IL-1β、IL-6的影响

目的:观察牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)上清液对体外培养的兔血管平滑肌细胞分泌IL-1β、IL-6的影响。方法:组织块法培养兔腹主动脉血管平滑肌细胞,并对其细胞来源进行鉴定。用4.3×106 CFU/mL的Pg上清液刺激细胞12、24、48 h后,通过ELISA检测IL-1β、IL-6的水平;同时用RT-PCR检测其mRNA表达的情况。结果:Pg上清液刺激24 h和48 h时能促进血管平滑肌细胞分泌IL-6,分别与同一时间点的对照组相比差异均有统计学意义(P<0.05),而且24 h时,IL-6表达最强,而IL-1β的表达最低,明显低于相同时间的对照组和其他时间点的实验组(P<0.05)。RT-PCR检测显示,4.3×106 CFU/mL的Pg上清刺激血管平滑细胞12、24、48 h后细胞内均有IL-1β、IL-6的基因表达,在24 h时,血管平滑肌细胞的IL-1β基因表达减少,而IL-6基因表达增加。结论:Pg上清液可促进细胞合成和分泌IL-6,在动脉粥样硬化的发生发展过程中可能发挥一定作用。     

牙龈卟啉单胞菌感染对血管内皮细胞与单核细胞黏附的影响

目的：研究Pg感染对人脐静脉内皮细胞（HUVEC）与单核细胞黏附的影响。方法：建立Pg感染HUVEC细胞株EA．hy926的体外模型,虎红染色法观察Pg381和Pg33277菌株感染HUVEC 6 h和24 h后,HUVEC与人单核细胞株THP-1细胞黏附量的变化。结果：培养6 h时Pg381感染组的THP-1细胞黏附量相对值为0．210±0．025,高于Pg33277感染组（0．078±0．024,P＜0．05）和空白对照组（0．062±0．022,P＜0．05）,Pg33277感染组和空白对照组无差异。24 h时3组间无差异（P＞0．05）。结论：Pg381感染HUVEC后能显著促进其与单核细胞的黏附。     

不同fimA基因型牙龈卟啉单胞菌对人脐静脉内皮细胞产生血管细胞黏附分子1和细胞间黏附分子1的影响

目的 观察不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)产生血管细胞黏附分子1(vascular cell adhesion molecule-1,VCAM-1)和细胞间黏附分子1(intercellular adhesion molecule-1,ICAM-1)的影响,探讨Pg在动脉粥样硬化发生、发展中的可能作用.方法 实验分别以PgATCC33277 (Ⅰ fimA ) 、WCSP115 (Ⅱ fimA)、W83 (Ⅳ fimA)和大肠杆菌脂多糖刺激HUVEC作为T1、T2、T3组(3个实验组)和阳性对照组,未受刺激的HUVEC作为阴性对照组;标准条件下厌氧培养上述3型Pg,将其以及大肠杆菌脂多糖分别与 HUVEC共同孵育2、6、24 h,采用流式细胞术检测HUVEC表面ICAM-1和VCAM-1的蛋白表达量,并通过激光共聚焦显微镜观察ICAM-1和VCAM-1的表达分布情况.结果 Ⅰ、Ⅱ、Ⅳ fimA型Pg刺激HUVEC后,细胞表面ICAM-1表达均增强(P<0.05),2、6、24 h表达量分别为Ⅰ fimA:60.27±5.43、80.81±1.44、85.94±2.56;Ⅱ fimA:86.69±8.81、90.19±0.00、96.18±0.48,Ⅳ fimA:59.66±0.40、85.79±4.86、96.04±2.07.除2 h时ⅠfimA与Ⅳ fimA型Pg刺激的HUVEC表面ICAM-1表达量差异无统计学意义外,其他各时间点Ⅱ、Ⅳ fimA型Pg的刺激作用均强于Ⅰ fimA型Pg(P<0.05).本研究条件下,Ⅰ、Ⅱ、Ⅳ fimA型Pg刺激HUVEC后2、6、24 h表达VCAM-1的水平均较低,各实验组与对照组间相比差异均无统计学意义(P>0.05).激光共聚焦显微镜观察显示,Pg刺激下HUVEC表达ICAM-1和VCAM-1增加,在Ⅱ、Ⅳ fimA型Pg刺激下,HUVEC中ICAM-1和VCAM-1荧光点相对较多且分布范围广.结论 牙周主要致病菌Pg毒力和致病性与其fimA基因型相关,Ⅱ fimA和Ⅳ fimA型Pg 有较强的上调HUVEC表达细胞黏附分子的能力,可能导致血管内皮功能紊乱.

Abstract：

Objective To investigate the effect of Porphyromonas gingivalis(Pg) with different fimA genotypes on vascular cell adhesion molecule-1(VCAM-1) and intercellular adhesion molecule-1(ICAM-1) production by human umbilical vein endothelial cells(HUVEC). Methods In the present study, PgATCC33277(type Ⅰ fimA genotype), WCSP 115(type Ⅱ fimA genotype), W83(type Ⅳ fimA genotype), and Escherichia coli-lipopolysaccharide (Ec-LPS) were designed as experimental group 1, 2, 3, and positive control group, respectively, to stimulate HUVEC, and the un-stimulated HUVEC were analyzed as negative control group. The three strains of Pg were cultured anaerobically in standard condition, and then the Pg cells and Ec-LPS were co-cultured with HUVEC for 2, 6, and 24 h, respectively. The amount of ICAM-1 and VCAM-1 produced by HUVEC was detected with flow cytometry(FCM). The expression of ICAM-1 and VCAM-1 by HUVEC were assayed with confocal laser scanning microscope(CLSM). ResultsThe expression of ICAM-1 on the surface of HUVEC were intensified after infected by Pg with Ⅰ, Ⅱ, and Ⅳ fimA genotypes (P<0.05). The amounts of ICAM-1 were 60.27±5.43, 80.81±1.44, and 85.94±2.56 for Pg with type Ⅰ fimA genotype, 86.69±8.81, 90.19±0.00, and 96.18±0.48 for Pg with type Ⅱ fimA genotype, 59.66±0.40, 85.79±4.86, and 96.04±2.07 for Pg with type Ⅳ fimA genotype at 2, 6 and 24 h after infection, respectively. The up-regulation effects caused by Pg with type Ⅱ and Ⅳ fimA genotypes were stronger than those caused by Pg with type Ⅰ fimA genotype at different time points except at 2 h(P<0.05). Under the present experimental condition, infected by Pg with type Ⅰ, Ⅱ and Ⅳ fimA genotypes stimulated low expression of VCAM-1 by HUVEC, it showed no significant differences among all the groups (P>0.05). Expression of ICAM-1 and VCAM-1 in Pg infected HUVEC were confirmed by CLSM. Infection of HUVEC with Pg resulted in more fluorescence staining of ICAM-1 and VCAM-1 compared with that in uninfected HUVEC cultures. Conclusions The virulence and pathogenicity of Pg is associated with its fimA genotypes, Pg with type Ⅱ and Ⅳ fimA genes possess stronger ability to stimulate HUVEC to up-regulate the expression of cell adhesion molecules, which may lead to disorders in vascular endothelial function.     

牙龈卟啉单胞菌对人脐静脉内皮细胞分泌IL-1β和IL-6的影响研究

目的:研究牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)上清液对人脐静脉内皮细胞(hu-man umbilical vein endothelial cells,HUVECs)分泌IL-1β、IL-6的影响。方法:酶消化法原代培养HUVECs,并对细胞进行来源鉴定,用5×106CFU/mL的Pg上清液刺激HUVECs 6、12、18、24 h后,RT-PCR检测IL-1β、IL-6 mRNA的表达;同时ELISA方法测定IL-1β及IL-6蛋白的水平。结果:Pg上清液刺激HUVECs后,IL-1βmRNA水平在12、18 h时蛋白明显高于对照组(P<0.05);IL-6 mRNA水平在12 h的表达明显高于对照组(P<0.05)。IL-1β蛋白在18、24 h的表达明显高于对照组(P<0.05);IL-6蛋白在12、18、24 h 3个时点均明显高于对照组(P<0.05)。结论:5×106CFU/mL Pg上清液可促进人脐静脉内皮细胞IL-1β和IL-6的基因及蛋白表达,提示Pg感染可能在As的发生发展中起一定作用。     

牙龈卟啉单胞菌对人滋养层细胞增殖及凋亡的影响

目的:检测牙龈卟啉单胞菌感染对人胎盘滋养层细胞(HTR-8细胞)增殖以及凋亡的影响。方法:用不同感染复数(MOI)的牙龈卟啉单胞菌体标准菌ATCC 33277体外感染HTR-8细胞,未感染牙龈卟啉单胞菌的HTR-8细胞作为空白对照组。感染后,用Cell Counting Kit(cck-8)检测HTR-8细胞增殖情况,用线粒体膜电位凋亡检测试剂(JC-1)检测HTR-8细胞凋亡情况。结果:MOI=10组,感染72 h内,HTR-8细胞增殖未受抑制(P>0.05)。而MOI=100组和MOI=200组,感染24 h开始,HTR-8细胞增殖受到明显抑制(P<0.01)。并且与MOI=100组相比,MOI=200组在感染48 h(P<0.05)和72 h(P<0.01)时,HTR-8细胞增殖明显降低。牙龈卟啉单胞菌感染24 h,MOI=200组HTR-8细胞凋亡率显著高于空白对照组(P<0.01)。感染48 h,MOI=100组HTR-8细胞凋亡率显著高于对照组(P<0.01)。MOI=10组在感染24 h和48 h时细胞凋亡率与空白对照组相比差异无统计学意义。结论:在体外感染模型中,牙龈卟啉单胞菌标准菌ATCC 33277感染可抑制滋养层细胞增殖,且促进其凋亡。提示早产低体重儿与牙龈卟啉单胞菌感染引起滋养层细胞增殖和凋亡失衡有关。     

牙龈卟啉单胞菌脂多糖对人脐静脉内皮细胞表达趋化因子RANTES和分形素的影响

目的 观察牙龈卟啉单胞菌脂多糖（Pg-LPS）对人脐静脉内皮细胞（HUVEC）表达调节活化正常T细胞表达和分泌的趋化因子（RANTES）和分形素的影响。方法 应用不同质量浓度（200、500、1 000 ng·mL^-1）的Pg-LPS分别处理HUVEC 1、6、12、24 h,利用实时荧光定量聚合酶链反应（RT-PCR）及酶联免疫吸附（ELISA）法检测RANTES及分形素mRNA及蛋白质的表达变化。结果 在Pg-LPS与HUVEC共同培养1、6和12 h时,除培养时间为12 h、Pg-LPS质量浓度为200 ng·mL^-1组的RANTES mRNA和1 h、200 ng·mL^-1组RANTES蛋白表达与对照组无明显差异外,其余各实验组RANTES蛋白和mRNA表达量及分形素mRNA表达量均高于对照组,差异有统计学意义（P<0.05）;6 h时,二者mRNA表达量达到峰值,分别为对照组的4.88倍和6.20倍;刺激6 h后,RANTES蛋白和mRNA表达量及分形素的mRNA表达量均降低,24 h时,仅Pg-LPS质量浓度为500 ng·mL-1组与对照组相比有统计学差异（P<0.05）;分形素蛋白的表达量则仅在Pg-LPS浓度为1 000 ng·mL-1刺激6、12 h时与对照组相比有统计学差异（P<0.05）。结论 Pg-LPS感染具有上调HUVEC表达趋化因子RANTES和分形素的作用,可能在牙周炎促进动脉粥样硬化发生、发展的过程中起一定作用。     

17-β雌二醇对牙龈卟啉单胞菌作用下人牙周膜细胞表达白细胞介素6、8的影响

目的 观察17-β雌二醇(estradiol,E2)对牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg) W83作用下人牙周膜细胞(human periodontal ligament cells,hPDLC)白细胞介素(interleukin,IL)6和IL-8表达的影响.方法 原代培养hPDLC,传至第4代,分别以①10-10 mol/L E2(E21组);②10-7 mol/L E2(E22组);③感染复数为10∶1的Pg( Pg1组);③感染复数为100∶1的Pg(Pg2组);⑤感染复数为100∶1的Pg+ 10-10 mol/L E2(Pg2 +E21组);⑥感染复数为100∶1的Pg+10-7 mol/LE2(Pg2+ E22组)处理hPDLC 12和24h;以0.1％无水乙醇为对照组,酶联免疫吸附测定法榆测细胞IL-6和IL-8蛋白的表达水平,实时荧光定量反转录聚合酶链法检测24 h mRNA水平.采用单因素方差分析比较各组IL-6、IL-8蛋白和mRNA表达水平的组间差异,最小显著性差异事后比较检验进行组间差异的两两比较,采用独立样本t检验分析各组12和24 h IL-6、IL-8表达水平的差异,检验水准为双侧α=0.05.结果 Pg2组24h时IL-6蛋白表达水平[(2482.88±26.53) ng/L]显著高于Pg1组[(734.09±87.90)ng/L]、对照组[(425.8±77.25) ng/L]和12 h时的Pg2组[(1157.50±234.65) ng/L,P=0.000];Pg2组24 h时IL-8蛋白表达水平[(4965.81±1072.55) ng/L]显著高于Pg1组[(803.51 ±162.08) ng/L,P=0.007]、对照组[(400.75±2.27) ng/L,p=0.005]和12h时的Pg2组[(1431.12 ±82.78)ng/L,P=0.001].E2对hPDLC IL-6和IL-8的表达无显著调节作用.与感染复数100∶1的Pg单独作用相比,E2和Pg联合处理24 h显著促进了hPDLC IL-6的表达水平,而未影响IL-8的表达水平:Pg2 +E21组、Pg2+ E22组IL-6 mRNA相对于磷酸甘油醛脱氢酶的表达水平(分别为0.49±0.15、0.53±0.16)显著高于Pg2组(0.19±0.06) (P =0.021,P=0.036),两组IL-6蛋白水平[分别为(5512.66±1022.07)、(6988.78±2279.13) ng/L]亦显著高于Pg2组[(3138.46±183.72) ng/L](P=0.012,P=0.000).结论 PgW83促进hPDLC IL-6和IL-8的表达呈剂量和时间依赖性;无牙周致病菌感染时E2对hPDLC IL-6和IL-8的表达无显著调节作用;而在牙周感染Pg时E2可能与Pg协同直接促进hPDLC产生IL-6,从而可能促进了牙周炎症过程.     

Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases

Sun W, Wu J, Lin L, Huang Y, Chen Q, Ji Y. Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases. J Periodont Res 2010; 45: 381–388. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: The purpose of this study was to examine the ability of Porphyromonas gingivalis to invade human umbilical vein endothelial cells (HUVECs) and to study the effects of P. gingivalis ATCC 33277 on the production of nitric oxide (NO) and on the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in HUVECs. We attempted to throw light on the pathway of damage to endothelial function induced by P. gingivalis ATCC 33277. Material and Methods: P. gingivalis ATCC 33277 was cultured anaerobically, and HUVECs were treated with P. gingivalis ATCC 33277 at multiplicities of infection of 1:10 or 1:100 for 4, 8, 12 and 24 h. HUVECs were observed using an inverted microscope and transmission electron microscopy. NO production was assayed through measuring the accumulation of nitrite in culture supernatants. Expression of both iNOS and eNOS proteins was investigated through western blotting. Results: It was found that P. gingivalis ATCC 33277 can adhere to HUVECs by fimbriae, invade into HUVECs and exist in the cytoplasm and vacuoles. P. gingivalis ATCC 33277 can induce iNOS and inhibit eNOS expression, and stimulate the release of NO without any additional stimulant. Conclusion: Our study provides evidence that P. gingivalis ATCC 33277 can invade HUVECs, and the ability of P. gingivalis ATCC 33277 to promote the production of NO may be important in endothelial dysfunction, suggesting that P. gingivalis ATCC 33277may be one of the pathogens responsible for atherosclerosis.     

NO production in RAW264 cells stimulated with Porphyromonas gingivalis extracellular vesicles

Oral Diseases (2010) 17 , 83–89 Objective: This experiment was carried out in order to prove the inducible nitric oxide synthase (iNOS) expression and the nitric oxide (NO) production in mouse macrophage cells (RAW264) which were stimulated by vesicles released from Porphyromonas gingivalis, and discussed about the role of vesicles in advance periodontal diseases. Materials and Methods: Production of NO₂^? in RAW264 cells was investigated after 0, 1, 3, 6 and 12 h of stimulation with P. gingivalis vesicles. NO was analyzed by HPLC‐based flow reactor system with Griess reagent. The cells stained by the enzyme‐labeled antibody method, after being stimulated with vesicles for 12 h. The iNOS proteins, which were expressed in RAW264 cells after 12 h of stimulation with vesicles, were detected by western blot. Results: When stimulated with vesicles from W83 and from ATCC33277, the RAW264 cells produced NO, but cell proteins that came in contact with the vesicles were degraded by protease activities in vesicles. When stimulated with vesicles from gingipain‐deficient mutant strain KDP136, the RAW264 cells produced NO, but the quality was about 60%, compared with the vesicles from ATCC33277. Conclusion: The results suggest that vesicles are not only just a part of bacterial component, but also are a toxic complex of lipopolysaccharide and protease, and one of the putative virulence factor for periodontal diseases that continue inflammation and cause chronic conditions.     

牙龈卟啉单胞菌感染血管平滑肌细胞的超微结构观察

目的:观察牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)对大鼠胸大动脉平滑肌细胞黏附及入侵情况,探讨Pg对血管平滑肌细胞的损伤途径,为牙周病在动脉粥样硬化发病机制中的研究提供依据.方法:用感染复数(multiplicity of infection,MOD为100∶1 Pg感染大鼠胸大动脉平滑肌细胞,分别于8、16 h TEM观察Pg对血管平滑肌细胞的黏附和入侵能力.结果:Pg感染大鼠胸大动脉平滑肌细胞16h时,Pg能利用表面菌毛黏附于血管平滑肌细胞表面并入侵胞质内.结论:Pg能够黏附于大鼠胸大动脉平滑肌细胞,并入侵细胞质,定植于细胞内.     

Outer membrane vesicles from Porphyromonas gingivalis affect the growth and function of cultured human gingival fibroblasts and umbilical vein endothelial cells

BACKGROUND: The purpose of this study was to examine the effects of outer membrane vesicles (OMV) obtained from Porphyromonas gingivalis (Pg) on the growth and function of human gingival fibroblasts (HGF) and human umbilical vein endothelial cells (HUVEC). METHODS: OMV were obtained from a cell-free growth medium of Pg ATCC 33277 by 40% NH2SO4 precipitation and ultracentrifugation. Cell proliferation was measured by 3H-thymidine incorporation into growing HGF and HUVEC. Endothelial cell function was determined by their capacity to form a network of capillary tubes on an extracellular matrix (ECM). RESULTS: Proliferating HGF and HUVEC demonstrated a significant dose-dependent inhibition of 3H-thymidine uptake when cultured with 0 to 40 microg/ml of OMV protein. HGF and HUVEC showed an IC50 of growth of about 9.0 microg/ml and 4.5 microg/ml of OMV protein, respectively. Capillary tube formation by HUVEC cultured on an ECM was suppressed by 70% to 80% with 5 microg/ml OMV protein after 18 hours of incubation. The presence of proteolytic enzymes in the OMV did not contribute to capillary tube disruption, since blocking enzyme activity with specific inhibitors did not reduce the suppression of capillary tube formation. After heating at 90 degrees C for 5 minutes, OMV significantly lost their capacity to suppress capillary tube formation. CONCLUSIONS: OMV significantly inhibit the proliferation of cultured HGF and HUVEC in a dose-dependent manner. OMV suppressed the capillary tube formation by cultured HUVEC. The factor(s) appeared to be a protein and not endotoxin because its inhibitory activity was markedly reduced by heat inactivation. These studies suggest that OMV contribute to chronic periodontitis by suppressing cell proliferation and revascularization in periodontal tissues.     

l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l ‐arginine and/or N^G‐monomethyl‐l ‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l ‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l ‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l ‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.     

牙龈卟啉单胞菌影响血管内皮细胞增殖和凋亡的实验研究

目的 观察牙龈卟啉单胞菌(Porphyromonas gingivatis,Pg)对血管内皮细胞增殖和凋亡的影响,探讨Pg在动脉粥样硬化发病机制中的作用.方法 建立体外Pg侵入血管内皮细胞模型,甲基噻唑基四唑(MTT)法观察细胞增殖,碘化丙啶染色、流式细胞仪分析细胞周期,Annexin-V-FITC凋亡试剂盒检测细胞凋亡.结果 PgATCC33277侵入后72 h细胞增殖活性降低12.46%,Pg W83侵入后72 h细胞增殖活性降低10.47%(F=786.68,P<0.01);Pg W83侵入后24 h使G1期细胞增加(F=43.23,P<0.01),ATCC 33277侵入后48 h使G1期细胞增加(F=66.72,P<0.01);Pg侵入后24 h即诱导细胞凋亡(F=1074.56,P<0.01).结论 Pg可能通过细胞毒性及诱导凋亡作用,加重血管内皮细胞的局部炎性反应,在动脉粥样硬化炎性病理反应中有重要意义.     

四种牙周致病菌侵入血管内皮细胞能力的体外研究

目的 研究和比较4种常见牙周致病菌Pg33277、Pi25611、Aa29522和Fn10953黏附和侵入人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的能力,为深入探讨牙周致病菌感染HUVEC在促进动脉粥样硬化发生、发展中的作用奠定初步基础.方法 建立牙周致病菌感染HUVEC的体外模型,采用扫描电镜、抗生素保护-菌落计数法观察以上牙周致病菌黏附和侵入HUVEC的能力.结果扫描电镜结果表明Pg33277、Pi25611、Aa29522和Fn10953均可黏附于HUVEC;抗生素保护法结果发现Pg33277、Pi25611、Aa29522和Fn10953均可侵入HUVEC,细菌侵入量分别为(0.8±0.1)×10~8、(4.1±0.5)×10~6、(1.6±0.3)×10~6、(5.0±0.4)×10~6CFU/L,侵入率分别为(0.400±0.050)%、(0.021±0.003)%、(0.008±0.002)%和(0.025±0.002)%,Pg33277侵入能力远较其他3种牙周致病菌强(P＜0.001),其余3种牙周致病菌间的侵入能力差异无统计学意义(P＞0.05).结论 4种常见牙周致病菌Pg33277、Pi25611、Aa29522和Fn10953均可黏附和侵入HUVEC,Pg33277侵入能力最强,这可能为其进一步发挥相关的生物学作用奠定基础.     

牙龈卟啉单胞菌对脐静脉血管内皮细胞表达IL-8和MCP-1 mRNA的影响

目的：在转录水平上观察牙龈卟啉单胞菌对脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVECs）表达白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）的影响。方法：厌氧培养Pg，原代培养HUVECs，RNA抽提，逆转录-聚合酶链式反应（RT-PCR）和mRNA比色定量法检测IL-8和MCP-1基因表达。结果：HUVECs基础表达IL-8和MCP-1mRNA,Pg以剂量依赖的方式增强IL-8和MCP-1mRNA的表达，Pg感染后1hIL-8mRNA开始增高，3h达到高峰，并持续到5h。而MCP-1的mRNA从Pg感染后1h开始增高，3h达到高峰，5h出现下降趋势。结论：Pg在转录水平上增强UHUVECs表达IL-8和MCP-1，这种上调作用可能是牙周病早期基因水平调控炎症细胞募集的重要因素，可能是牙周疾病的炎症反应和免疫反应中主要调控机制之一。