牙龈卟啉单胞菌入侵血管内皮细胞的实验研究

Porphyromonas Gingivalis Invasion of Vein Endothelial Cells

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。

Objective： To examine the ability of Porphyromonas gingivalis ATCC33277 （Pg ATCC33277） to invade human umbilical vein endothelial cells（HUVEC） and to observe the effect of Pg ATCC33277 on the proliferation of HUVEC in vitro. Methods： Pg ATCC 33277 was cultured anaerobically, and HUVEC were treated with Pg ATCC 33277 at multiplicities of infection （MOI） of 1 ：10 or 1：100 for 4, 8, 12 and 24 h. HUVEC were observed using an inverted microscope and transmission electron microscopy and the CCK-8 test was used to assay the proliferation of HUVEC. Results： Compared with uninfected controls, HUVEC co-incubated with Pg ATCC 33277 at MOI of 1： 10 or 1：100 respectively were present as an intact monolayer in 24 h. It was found that Pg ATCC 33277 could invade into HUVEC and exist in the cytoplasm and vacuoles. The proliferation of HUVEC expressed no significant difference in the groups co-cultured with Pg ATCC 33277and the control group. Conclusion.. Our study provides evidence that Pg ATCC 33277 can invade in 24 hHUVEC, and the Morphology and proliferation of HUVEC express no significant difference in the groups co-cultured with Pg and the control group. It has the capacity to persist within this host cell and affords them protection from the host immune system, which forms a second chronic infection process and may affect endothelial cell function.