Ⅰ、ⅣfimA型牙龈卟啉单胞菌对内皮细胞与平滑肌细胞共培养体系产生内皮素-1及一氧化氮的影响

The effect of Ⅰ and ⅣfimA genotypes of porphyromonas gingivalis on the production of endothelin-1 and nitric oxide by cocultured human umbilical vein endothelial cells with human artery smooth muscle cells

目的研究不同fim A基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)刺激人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)及人脐动脉血管平滑肌细胞(human umbilical artery smooth muscle cells,HUASMCs)共培养体系产生内皮素-1(endothelin-1,ET-1)和一氧化氮(NO)的水平。方法用Ⅰfim A型P.gingivalis(ATCC33277)和Ⅳfim A型P.gingivalis(W83)分别刺激HUVECs-HUASMCs共培养体系,于2、8、24、48 h时收集细胞培养上清液,酶联免疫反应检测ET-1的含量,硝酸还原酶法测定NO的含量。各组均设阴性对照组(纯培养基)及阳性对照组(1μg/m L E.coli-LPS)。结果 HUVECs-HUASMCs共培养体系在Ⅰ、Ⅳfim A型P.gingivalis刺激作用下产生ET-1和NO的量以及ET-1/NO水平,与阴性及阳性对照组比较存在差异。Ⅰfim A型P.gingivalis刺激细胞共培养模型分泌ET-1和NO情况的总趋势与阴性对照组相似、而Ⅳfim A型P.gingivalis的刺激作用总趋势则与阳性对照组相似,Ⅳfim A型P.gingivalis较Ⅰfim A型P.gingivalis刺激共培养细胞可分泌更多的ET-1、而NO量减少,Ⅳfim A型P.gingivalis感染48 h的细胞共培养模型表现出明显的ET-1/NO的失衡。结论不同fim A型P.gingivalis刺激共培养模型后产生ET-1及NO的情况及ET-1/NO的水平有明显差异,可能与其本身毒力相关,Ⅳfim A型P.gingivalis比Ⅰfim A型P.gingivalis更易引起内皮功能紊乱。

Objective To observe the effects of different fim A genotypes of Porphyromonas gingivalis（ P. gingivalis） on the production of endothelin-1（ ET-1） and nitric oxide（ NO） by co-cultured human umbilical vein endothelial cells（ HUVECs） with human umbilical artery smooth muscle cells（ HUASMCs）. Methods P. gingivalis ATCC33277（ type Ⅰ fim A gene） and W83（ type Ⅳ fim A gene） were cultured anaerobically in standard condition,and a novel co-culture system of HUVECs and HUASMCs was treated with different fim A genotypes of P. gingivalis for 2,8,24 and 48 h. At different time points,the supernatant was collected,the levels of ET-1 were determined by ELISA,and the levels of NO were determined by nitrate reductase. A negative control group（ blank control） and a positive control group（ 1ug / m L E. coli- LPS） were set in each experimental group. Results The co-culture system of HUVECs and HUASMCs produced ET-1 and NO with Ⅰand Ⅳfim A genotypes of P. gingivalis stimulation. Compared with the negative and positive control groups,differences were observed concerning the ET-1 and NO production and ET-1 / NO level in the experimental group. In terms of overall trend of the production of ET-1 and NO,the group ofⅠ fim A genotype of P. gingivalis was similar to the negative control group,while the group of Ⅳfim A genotype of P. gingivalis was similar to the positive control group. Ⅳfim A genotype of P. gingivalis demonstrated more secretion of ET-1 and a lower amount of NO compared toⅠ fim A genotype of P. gingivalis. At 48 h,co-cultured HUVECs and HUASMCs infected by Ⅳfim A genotype of P. gingivalis showed a significant imbalance of ET-1 / NO. Conclusions Stimulated by different fim A genotypes of P. gingivalis,the production of ET-1 and NO by co-cultured HUVECs and HUASMCs,and the ET-1 / NO level were significantly different between two fim A genotype of P. gingivalis,which may be related to the native virulence of the bacteria. Ⅳfim A genotype of P. gingivalis could stimulate and influence co-cultured HUVECs and HUASMCs,and is more likely to cause endothelial dysfunction thanⅠ fim A genotype P. gingivalis.