慢性牙周炎口腔变黑普雷沃氏菌分离鉴定及促进食管鳞癌演进

Isolation and identification of Prevotella nigrescens in patients with chronic periodontitis and analysis of its tumorigenic role in esophageal squamous carcinogenesis

目的分离、鉴定口腔变黑普雷沃氏菌(Prevotella nigrescens,P.nigrescens),探讨其对食管鳞癌是否有促进作用。方法胰豆胨肝粉琼脂(GAM)培养基厌氧原代培养慢性牙周炎患者龈沟液,挑选黑色菌落分离纯化;革兰染色、16S rRNA基因测序鉴定分离的单克隆细菌;该细菌感染食管鳞癌细胞NE6-T后细胞生物学性状变化。结果慢性牙周炎患者龈沟液厌氧原代培养120 h后可见多个菌落,其中灰黑色菌落经连续划线法分离纯化为黑色、圆形光滑单克隆菌落。该细菌革兰染色阴性、串珠状排列,16S rDNA序列与P.nigrescens F0103同源性为99.78%(P.nigrescens LY01)。LY01感染促进NE6-T细胞体外增殖、迁移、侵袭和裸鼠皮下荷瘤生长,并诱导Ki67表达上调和p-STAT3激活。结论慢性牙周炎口腔P.nigrescens可能促进了食管鳞癌发展。

Objective To isolate and identify Prevotella nigrescens(P.nigrescens)in gingival crevicular fluid of patients with chronic periodontitis and to analyze its tumor-promoting role in esophageal squamous cell carcinoma(ESCC).Methods Samples of gingival crevicular fluid were collected from patients with chronic periodontitis and cultured on GAM agar medium under anaerobic conditions.Black colonies on GAM agar plates were picked for subculture,and then the bacteria were isolated and purified.Bacterial identification was conducted using Gram staining and 16S rDNA sequencing analysis.The isolated bacterial species was used to infect ESCC NE6-T cells and the changes in biological characteristics of the infected cells were evaluated.Results Multiple bacterial colonies were observed after anaerobic culturing of gingival crevicular fluid samples for 120 h on GAM agar plates.A single bacterial colony with pure black and smooth appearance was obtained from grey black bacterial colonies with streak method.It was a Gram-negative bacterium with bead-like shape.It showed 99.78%16S rDNA sequence identity with P.nigrescens F0103 and was named P.nigrescens LY01.P.nigrescens LY01 infection promoted the in vitro proliferation,migration and invasion of NE6-T cells and the growth of subcutaneous xenograft in nude mice.In addition,it could also induce the upregulation of Ki67 and activation of p-STAT3.Conclusions P.nigrescens inhabiting in gingival sulcus might promote the progression of ESCC in human with chronic periodontitis.