人血管内皮细胞的体外培养

目的:观察体外培养的血管内皮细胞的生物学特性.方法:应用酶消化法获取人主动脉和脐静脉血管内皮细胞,体外培养并传代;观察细胞形态、测定生长曲线和培养液中6-酮-PGF1α的分泌量,应用免疫组化法对其进行鉴定,并对两种来源内皮细胞的生物学特性进行比较.结果:应用酶消化法成功获取了人主动脉和脐静脉血管内皮细胞.两种来源的内皮细胞细胞形态相仿,均具有较强的生长增殖能力,在细胞对数生长期分泌PGI2的能力旺盛.结论:人主动脉和脐静脉均是良好的血管内皮细胞来源材料,适于体外培养,二者的生物学特性相似.     

牙周可疑致病菌对胶原包被羟磷灰石黏附的体外实验研究

目的比较对牙根面具有黏附能力的具核梭杆菌、牙龈卟啉单胞菌、中间普氏菌和伴放线菌嗜血菌对胶原包被的羟磷灰石实验膜（C- HA）的黏附能力,初步探讨以上牙周可疑致病菌在牙根表面形成菌斑生物膜的能力。方法采用同位素闪烁计数法测定上述4种细菌黏附至C- HA表面的黏附量及黏附率,比较其黏附能力。结果培养相同时间,不论是培养24 h还是培养48 h,4种不同细菌两两比较,具核梭杆菌ATCC 10953和牙龈卟啉单胞菌ATCC 33277对C- HA表面黏附率的差异无统计学意义;中间普氏菌ATCC 25611和伴放线菌嗜血菌ATCC 29523的黏附率之间差异也无统计学意义,但是具核梭杆菌ATCC 10953和牙龈卟啉单胞菌ATCC 33277对C- HA表面的黏附率显著高于中间普氏菌ATCC 25611和伴放线菌嗜血菌ATCC 29523（P<0.001）。同一种细菌,在培养不同时间即培养24 h和48 h,对C- HA表面黏附率的差异均无统计学意义。结论不同的牙周可疑致病菌对胶原包被的羟磷灰石的选择性黏附作用不同,具核梭杆菌和牙龈卟啉单胞菌对胶原有较强的亲和作用,在细菌的局部定植过程和牙周炎的进展和复发中可能发挥重要作用。     

牙周致病菌内毒素耐受的研究进展

内毒素耐受(endotoxin tolerance)是指先前的脂多糖(lipopolysaccharide,LPS)刺激使机体或体外培养的细胞对后续刺激反应性降低的一种现象,可能导致炎症因子释放的减少,进而在牙周炎发生发展过程中发挥重要作用。本文就牙周致病菌内毒素耐受的作用和机制的研究进展作一综述。     

口臭与牙周致病菌关系的研究

目的 探讨牙周致病菌与口臭之间的关系。方法 抽取100例不同梯度口气值的受试者,用N-苯甲酰-DL-精氨酰-2萘胺(BNA)试纸检测口臭患者牙周菌斑及舌背覆盖物中的牙周致病菌。将每位受试者的嗅觉评分、挥发性硫化物(VSC)水平与BANA实验的结果做相关分析。结果 VSC值和嗅觉评分与3处菌斑中BNA实验的结果均有显著性相关。结论 口臭与牙周袋及舌背覆盖物中的BANA活性细菌有关。口臭与牙周病的活动性有关。     

牙周可疑致病菌的传播性研究进展

现代分子生物学的快速进展为微生物的分类学研究提供了多种高敏感度的方法,使研究各类细菌的传播途径成为可能。近年来研究表明牙周可疑致病菌可交互传播,本文着重就其中与牙周疾病关系密切的相关细菌在人群中传播的可能性及途径的研究进展进行综述。     

动脉粥样硬化斑块中牙周致病菌的检测

目的:探讨牙周炎的程度是否影响动脉粥样硬化斑块中牙周致病菌的检出率.方法:29例患有不同程度牙周炎患者,行冠状动脉搭桥手术时,取动脉粥样硬化斑块及术前取龄下菌斑,采用chelex-100法提取细菌DNA,以聚合酶链反应法检测牙龄卟啉单胞菌、伴放线放线杆菌、福赛斯坦氏菌3种牙周炎相关致病菌.29人分为2组,重度牙周炎组1...     

成骨细胞与血管内皮细胞复合PDLLA的体外实验研究

目的：探讨兔成骨细胞与血管内皮细胞体外复合外消旋聚乳酸（PDLLA）的可能性,为血管化组织工程骨构建打下实验基础.方法：制备PDLLA浸提液培养血管内皮细胞,MTT法检测细胞活性.将制备好的PDLLA预湿、Ⅰ型胶原包埋;体外培养兔成骨细胞及血管内皮细胞后,与PDLLA复合10 d,扫描电子显微镜观察其在支架上的生长情况.结果：PDLLA浸提液对血管内皮细胞无毒性,成骨细胞与血管内皮细胞在支架上成片状分布,可见细胞外基质分泌.结论：两种细胞在经Ⅰ型胶原包埋的PDLLA上生长状态良好,可用于血管化组织工程骨的构建.     

牙周致病菌在动脉粥样硬化发生发展中的作用研究

牙周炎与全身健康特别是心血管疾病关系密切,牙周致病菌积极参与了动脉粥样硬化的形成与发展。目前已有研究证据表明,牙周致病菌感染是心血管事件的独立危险因素,其通过多种免疫炎症以及代谢相关分子机制促进动脉粥样硬化病变的发生发展。本文从牙周炎及牙周致病菌与心血管疾病的相关性、牙周致病菌影响动脉粥样硬化的机制等方面,对这一主题进...     

血管内皮细胞对骨髓间充质干细胞成骨能力影响的体外研究

目的研究血管内皮细胞对骨髓间充质干细胞成骨能力的影响.方法体外分离培养大鼠骨髓间充质干细胞和肾血管内皮细胞,三维培养体系中直接、间接接触培养后,检测细胞中蛋白质和骨钙素含量及碱性磷酸酶活性.结果直接、间接接触培养及单独培养的骨髓间充质干细胞蛋白质含量为(0.195±0.023) mg/mL、(0.174±0.015) mg/mL、(0.152±0.015) mg/mL、(0.141±0.014) mg/mL;碱性磷酸酶活性分别为(0.625±0.223) μ/mg、(0.412±0.178) μ/mg、(0.141±0.08) μ/mg ;骨钙素含量分别为(0.800±0.124) μg/L、(0.435±0.216) μg/L、(0.235±0.146) μg/L.经方差分析各组差异显著(P<0.05).结论骨髓间充质干细胞与血管内皮细胞接触培养有良好的细胞相容性,血管内皮细胞可以显著提高骨髓间充质干细胞的增殖能力和成骨能力.     

硫炔丙基半胱氨酸对三种牙周致病菌抗菌作用

目的:研究硫炔丙基半胱氨酸(S-propargyl-cysteine,SPRC)溶液对牙周炎常见致病菌的体外抗菌作用.方法:体外分别将牙龈卟啉单胞菌(P.gingivalis),中间普氏菌(P.intermedia)和粪肠球菌(E.faecalis)与不同浓度SPRC(10,20,50和100 mmol/L)共培养,没...     

Relatively low invasive capacity of Porphyromonas gingivalis strains into human gingival fibroblasts in vitro

ObjectiveBacterial invasion into host cells is a common strategy to escape the host immune system. Gingival fibroblasts (GFs) are the most predominant non-phagocytic cell type in gingival connective tissue. Therefore, invasion into GFs was thought to be the first strategy for the survival of Porphyromonas gingivalis. The present study compared the invasive ability of P. gingivalis into GFs with those of other red-complex and relatively less pathogenic bacterial strains, especially Fusobacterium nucleatum.Materials and methodsInvasive ability of bacterial strains into GFs was measured using a flow cytometric invasion assay at a multiplicity of infection of 1000. The effect of dual infection with F. nucleatum CCUG 37843T on P. gingivalis ATCC 49417 invasion was investigated. The invasive ability of F. nucleatum and P. gingivalis was confirmed using confocal microscopy.ResultsThe invasive ability of red-complex bacteria was markedly lower than that of F. nucleatum or Campylobacter gracilis. The invasive ability of 4 types and 10 clinical strains of P. gingivalis was less than 6%, and that of F. nucleatum strains was greater than 45%. Confocal analysis revealed that the percentage of bacteria invading GFs in the cell-treated P. gingivalis and F. nucleatum were 0.0068% and 1.22%, respectively. Dual infection with F. nucleatum increased the invasive ability of P. gingivalis.ConclusionThe invasive capacities of P. gingivalis into GFs were comparatively lower than those of relatively less pathogenic bacteria. Invasion into GFs cannot be the first strategy for survival of P. gingivalis in gingival connective tissue.     

Invasion of vascular cells in vitro by Porphyromonas endodontalis

AIM: The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. METHODOLOGY: Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. RESULTS: Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. DISCUSSION: Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.     

Ultrastructural morphology of vascular endothelial junctions in periodontal ligament

The TEM was used to categorize vessels and their junctions in normal and tensioned rat maxillary molar periodontal ligament. In tensioned periodontal ligament mean luminal diameters of capillaries were significantly smaller (p< 0.001). Goniometer tilting of sections with apparent tight regions revealed that only 16 per cent were actual tight junctions. The other regions proved to be close junctions (85 per cent) and open junctions (4 per cent). No gap junctions were found. These findings establish that morphologically the periodontal ligament contains a microvascular bed of 'leaky' endothelium with a potentially high permeability factor.     

人牙周膜干细胞体外诱导分化为神经元样细胞的实验研究

目的体外定向诱导人牙周膜干细胞分化为神经元样细胞,探讨人牙周膜干细胞的多向分化潜能。方法体外分离、培养人牙周膜细胞,待细胞达一定量后用有限稀释法进行克隆化培养,筛选鉴定牙周膜干细胞（PDLSC）,用含10 μg/L碱性成纤维细胞生长因子（bFGF）的预诱导培养液诱导培养24 h,然后换用含5 mmol/L β-巯基乙醇（β-ME）的诱导培养液诱导培养6 h,光镜下观察诱导细胞形态学的改变,免疫荧光、RT-PCR等方法检测鼠抗人神经元特异性烯醇化酶（NSE）、神经丝蛋白（NF）和胶质纤维酸性蛋白（GFAP）的表达。同时以未诱导细胞为对照。结果体外诱导培养6 h细胞即发生形态学改变,可看到典型的神经元样细胞;免疫荧光、RT-PCR检测诱导细胞表达神经元细胞特异性标志NSE、NF,未诱导细胞无表达。结论人牙周膜干细胞在体外可以诱导分化为神经元样细胞,具有多向分化的潜能。     

正畸牙移动中牙周血管内皮祖细胞在骨改建中的作用机制探讨

目的 探讨正畸牙移动中牙周血管内皮祖细胞在骨改建中的作用机制。方法 首先建立实验性大鼠牙移动模型,分离和培养血管内皮祖细胞(endothelial progenitor cells EPCs),用10 μmol/L Brdu标记EPCs并通过尾静脉注入模型大鼠,观察EPCs在牙周组织的分布情况。将VEGF加入EPCs细胞培养液,MTT法测细胞增殖能力,显微镜下观察细胞黏附情况,Transwell实验观察细胞迁移能力。制作不同时间点模型大鼠的VEGF免疫组织化学染色切片,与显微镜下观察不同时间点牙周组织VEGF的表达情况。采用SPSS 20.0软件包对数据进行统计学分析。结果 成功建立了大鼠牙移动模型,从心脏血中分离EPCs,显微镜下观察到有些为梭形,将Brdu标记的EPCs经尾静脉注入模型大鼠中,观察到随着时间的增加,荧光强度逐渐增加,在3 d的标本中荧光强度达到最强。各个时间点上颌第一、二磨牙之间的间隙均为实验组低于对照组,且具有显著性差异（P<0.05）。VEGF免疫组织化学染色结果显示,无论是张力侧还是压力侧,实验组表达量均高于对照组。在成骨细胞和破骨细胞,VEGF均有表达,14 d时达到最大值。EPCs增殖、黏附能力实验表明,VEGF促进EPCs增殖,使其黏附力增强。Transwell实验表明,VEGF促进EPCs的趋化作用,VEGF对EPCs的生物作用具有调控作用。结论 EPCs能够聚集于牙周组织,参与牙周组织骨改建。EPCs趋化到牙周组织后,通过VEGF等因子的相互调控作用,参与牙周组织的改建程,促进牙周组织修复和骨改建。     

Experimental periodontitis promotes transient vascular inflammation and endothelial dysfunction

Objectives

This study aimed to evaluate the systemic inflammatory response and cardiovascular changes induced by experimental periodontitis in rats.

Design

Experimental periodontitis was induced by placing a cotton ligature around the cervix of both sides of mandibular first molars and maxillary second molars in each male rat. Sham-operated rats had the ligature removed immediately after the procedure. Seven, 14 or 28 days after procedure, the effects of acetylcholine, sodium nitroprusside and phenylephrine were evaluated on blood pressure, aortic rings and isolated and perfused mesenteric bed. The blood was obtained for plasma Interleukin-6 (IL-6), C-reactive protein (CRP) and lipid evaluation. The mesenteric vessels were obtained to evaluate superoxide production and nitric oxide synthase 3 (NOS-3) expression.

Results

Ligature induced periodontitis reduced endothelium-dependent vasodilatation, a hallmark of endothelial dysfunction. This effect was associated with an increase in systemic inflammatory markers (IL-6 and CRP), worsens on lipid profile, increased vascular superoxide production and reduced NOS-3 expression. It is interesting to note that many of these effects were transitory.

Conclusion

Periodontitis induced a transient systemic and vascular inflammation which leads to endothelial dysfunction, an initial step for cardiovascular diseases. Moreover, the animal model of periodontitis used here may represent a valuable tool for studying the relationship between periodontitis and endothelial dysfunction.     

冠状动脉粥样硬化斑块中牙周炎致病菌的检测

目的探讨牙周炎与冠状动脉粥样硬化性心脏病（以下简称冠心病）发病的相关机制。方法收集31例行冠状动脉搭桥手术并伴有牙周炎的患者冠状动脉粥样硬化斑块和龈下菌斑，采用Chelex-100法提取细菌DNA，以聚合酶链反应（PCR）法检测冠状动脉粥样硬化斑块和龈下菌斑中牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）、伴放线放线杆菌（Actinobacillus actinomycetemcomitans，Aa）、具核梭杆菌（Fusobacterium nucleatum，Fn）、中间普氏菌（Prevotella intermedia，Pi）、福赛拟杆菌（Bacteroides forsythus，Bf）5种牙周炎相关致病菌。结果在31例患者冠状动脉粥样硬化斑块中，各种牙周炎相关致病菌的检出率分别为：Pg 38．7％、Aa 0％、Pi 12．9％、Fn 22．6％、Bf 38．7％；在本组同一患者冠状动脉粥样硬化斑块及龈下菌斑中同时检出Pg 5例、Aa 0例、Pi 2例、Fn 4例、Bf 8例，一致率分别为Pg 16．1％、Aa 0％、Pi 6．5％、Fn 12．9％、Bf 25．8％。结论在冠状动脉粥样硬化斑块中牙周炎相关致病菌DNA的检出，提示牙周炎相关致病菌在冠心病的发生、发展中可能起着重要的作用，为冠心病危险因素的研究和冠心病防治策略的制定提供了参考依据。     

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.     

Increased expression of C-reactive protein gene in inflamed gingival tissues could be derived from endothelial cells stimulated with interleukin-6

Background

Epidemiological studies have suggested periodontitis as a risk factor for ischemic heart disease. High sensitive C-reactive protein (hs-CRP), a predictor of cardiovascular risk, is elevated in periodontitis patients. Therefore, local infection-induced elevation of systemic CRP could account for the relationship between the 2 diseases. However, the underlying mechanism of CRP production in the periodontal tissues has not been fully elucidated. Therefore, the aim of the present study was to clarify the mechanism of CRP production in periodontal tissues.

Methods

Gene expression of CRP in gingival biopsies was analysed by quantitative PCR. Human gingival epithelial cells (HGECs), human gingival fibroblasts (HGFBs), and human coronary artery endothelial cells (HCAECs) were characterized for CRP-producing ability by incubating with interleukin (IL)-1β, IL-6, soluble IL-6 receptor (sIL-6R), and Porphyromonas gingivalis strain W83.

Results

Gene expression of CRP is significantly elevated in periodontitis lesions compared with gingivitis lesions. HCAECs, but not HGECs and HGFBs, produced CRP in response to IL-6 and IL-1β in the presence of sIL-6R. In contrast to IL-6, the effect of IL-1β on CRP production was indirect via induction of IL-6. IL-1β was produced by HGECs and HGFBs with stimulation of P. gingivalis antigens.

Conclusion

These results suggest that CRP induced locally by periodontal infection may play another role in the pathogenesis of periodontal disease, and to a much lesser extent, has the potential to modulate systemic CRP level by extra-hepatic CRP production.     

血管内皮细胞对牙周组织细胞增殖的影响

目的：通过血管内皮细胞与牙周组织细胞复合培养模拟牙周组织再生环境,观察血管内皮细胞对牙周组织细胞增殖的作用规律,探讨血管内皮细胞对牙周组织修复再生的影响。方法：应用原代培养的血管内皮细胞,在Transwell嵌套中实现与人牙周膜成纤维细胞、牙龈成纤维细胞间的复合培养,分别于复合培养第2、4、6、8和10天,以细胞计数手段检测血管内皮细胞共存条件下2种牙周组织细胞的增殖情况,并与单独培养的2种牙周组织细胞作为对照,采用SPSS13.0软件包对数据进行统计学分析。结果：在血管内皮细胞存在条件下,2种牙周组织细胞的增殖速率均显著高于单独培养条件。血管内皮细胞存在时,牙周膜成纤维细胞增殖速率逐渐超越牙龈成纤维细胞(第6天起),并在第8天细胞数量上超过牙龈成纤维细胞,差异具有显著性(P<0.01)。结论：血管内皮细胞的存在,对牙周组织细胞的增殖具有促进作用,对牙周膜成纤维细胞增殖的促进作用显著高于牙龈成纤维细胞。