目的比较不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,PS)刺激下人牙龈成纤维细胞单核细胞趋化蛋白1mRNA和蛋白的表达水平,比较不同fimA基因型Pg毒力和致病性的差异。方法PsATCC33277(Ⅰ型,T1组),WCSP115(Ⅱ型,他组),WCSP1.5(Ⅲ型,T3组),W83(Ⅳ型,T4组)分别与牙龈成纤维细胞在标准条件下共同孵育,对照组加入2ml达尔伯克氏改良伊格尔培养基,每组3孔。分别于孵育后1、3、6和12h收集细胞和上清液,应用实时荧光定量反转录聚合酶链反应(RT-PCR)和ELISA法分别检测牙龈成纤维细胞单核细胞趋化蛋白1mRNA和蛋白的动态表达。结果与对照组比较,各fimA型Pg刺激下牙龈成纤维细胞单核细胞趋化蛋白1mRNA和蛋白水平的表达量均明显上调(P〈0.05);其中他组Pg的刺激作用强于其他各型,不同时间点单核细胞趋化蛋白1mRNA相对表达量分别为(25.75±3.12)~(326.69±35.35),蛋白分泌水平为(178.20±46.20)~(443.46±82.19)ng/L;而乃组Pg弱于其他各型,单核细胞趋化蛋白1mRNA相对表达量为(4.16±0.82)~(94.17±18.56),蛋白分泌水平为(86.95±23.90)~(264.01±28.59)ng/L,差异有统计学意义(P〈0.05)。结论Pg可诱导牙龈成纤维细胞mRNA和蛋白水平过表达单核细胞趋化蛋白1,提示fimA基因型的不同可能与Pg的毒力和致病性存在相关性。
Objective To investigate the mechanism of monocyte chemoattractant protein-1 ( MCP-1 ) regulations of human gingival fibroblasts (HGF) by challenge of Porphyromonas gingivalis (Pg) with different fimA genotypes. Methods Pg ATCC 33277 ( type Ⅰ ), WCSP115 ( type Ⅱ ), WCSP1.5 ( type Ⅱ ), W83 ( type Ⅳ) were assessed for their inductions of MCP-1 expression in HGF. MCP-1 mRNA levels of HGF were determined by real-time RT-PCR and MCP-1 protein levels in culture supernatant by ELISA at different time intervals ( 1h, 3h, 6h and 12h) following continuous co-culture of bacteria with HGF. Results MCP-1 mRNA and protein levels were both up-regulated when HGF co-cultured with different Pg fimA genotypes. Type I1 was stronger than other fimA genotypes, HGF expressed significantly great amount of MCP-1 mRNA [ ( 25.75 ± 3. 12 ) -( 326. 69 ± 35. 35 ) ] and protein [ ( 178.20 ± 46. 20 )- (443.46 ±82. 19) ng/L] for different time periods; While Type Ⅲ was weaker than other fimA genotypes, and the level of MCP-1 mRNA was [ (4.16 ± 0.82)-(94.17 ± 18.56) ] and protein [ (86.95 ± 23.90)-(264.01 ± 28.59) ng/L ] (P〈0.05). Conclusions fimA genotypes of Pg are related with the inductions of MCP-1, which might indicate fimA genotype is associated with pathogenesis of Pg.