摘要目的:探讨口腔黏膜癌变过程中组织内细菌群落的差异，分析高丰度菌种中间普雷沃菌（Pi）与口腔黏膜癌变发生发展的关系。方法:收集2022年1月至2024年11月于南京大学医学院附属口腔医院就诊的口腔白斑病（OLK）、口腔鳞状细胞癌（OSCC）患者及口腔健康个体（HC）的新鲜组织样本，提取细菌的总DNA，利用2bRAD-M简化微生物组测序技术检测组织内菌群，并对OLK、OSCC和HC组的α、β多样性及物种组成进行分析和比较，以筛选出与癌变相关的细菌。随后，进一步收集临床石蜡组织，其中OLK组15例（单纯增生4例、轻度异常增生6例、中重度异常增生5例），OSCC组12例，HC组5例。同时，构建4-硝基喹啉N-氧化物（4NQO）诱导的OLK衍进小鼠模型，采用随机数字表法将小鼠随机分为3组，每组6只。阴性对照组饮用纯净水；实验组1，饮用含4NQO纯净水至12周；实验组2，饮用含4NQO纯净水至22周，小鼠处死后取舌体组织固定。应用特异性探针对人和小鼠组织切片内Pi 进行荧光原位杂交（FISH）验证，比较Pi的荧光强度和面积的差异，分析Pi的侵袭深度与组织分化程度的相关性。结果:2bRAD-M菌群分析结果显示，OSCC组织内Pi的相对丰度（10.80%）显著高于HC组（0.50%）（ P=0.001）和OLK组（0.70%）（ P=0.002）。Pi FISH检测结果显示，人OSCC组织内荧光强度（125.00±25.13）显著高于HC组（11.40±25.49）、单纯增生OLK组（11.75±23.50）和轻度异常增生OLK组（26.83±29.51）（ P=0.002， P=0.003， P=0.005），但与中重度异常增生OLK组（47.40±26.88）相比差异无统计学意义（ P=0.210）。OSCC组织内Pi荧光面积（9 255.00±2 048.00）显著高于HC组（18.00±40.25）、单纯增生OLK组（27.00±54.00）、轻度异常增生OLK组（76.00±95.19）和中重度异常增生OLK组（1 628.00±1 265.00）（均 P<0.001）。Pi侵袭深度与组织病理分级程度之间具有显著相关性（ P<0.001）。小鼠的OSCC组织中Pi的荧光强度（119.00±8.54）显著高于HC组（8.17±20.00）（ P<0.01），但与OLK组（34.33±28.28）相比差异无统计学意义（ P>0.05）。小鼠OSCC组织内Pi的荧光面积（9 971.0±2 807.0）显著高于HC组（37.83±92.67）和OLK组（451.6±780.1）（ P=0.006， P=0.043），Pi侵袭深度与组织病理分级程度之间具有显著相关性（ P<0.001）。 结论:本研究结果表明，口腔黏膜组织内Pi有作为早期检测OSCC生物标志物的潜力，Pi在口腔黏膜癌变过程中可能发挥重要作用。

abstractsObjective:To explore the differences in bacterial communities within tissues during the process of oral mucosal carcinogenesis, and analyze the relationship between the high-abundance species Prevotella intermedia (Pi) and the occurrence and development of oral mucosal carcinogenesis. Methods:Fresh tissue samples were collected from patients diagnosed with oral leukoplakia (OLK), oral squamous cell carcinoma (OSCC), and healthy controls (HC) at Nanjing Stomatological Hospital from January 2022 to November 2024, following strict inclusion criteria. Bacterial DNA was extracted from these specimens, and the 2bRAD sequencing for microbiome (2bRAD-M) was employed to analyze and compare the α and β diversity, as well as the community composition of bacteria within tissues, aiming to identify specifically expressed bacteria. Subsequently, paraffin-embedded clinical specimens were collected: 15 cases in the OLK group (including 4 cases of simple hyperplasia, 6 cases of mild dysplasia, and 5 cases of moderate to severe dysplasia), 12 cases in the OSCC group, and 5 cases in the HC group. A 4-nitroquinoline n-oxide (4NQO)-induced OLK progression mouse model was also constructed. Mice were randomly divided into three groups using a random number table, with six in each group. The negative control group was given distilled water to drink; Group 1 was given distilled water containing 4NQO to drink until week 12, while Group 2 was given distilled water containing 4NQO to drink until week 22. After the mice were sacrificed, their tongue tissue were collected and fixed. Fluorescence in situ hybridization (FISH) with specific probes was used to validate the presence of Pi in human and mouse tissue sections, analyzing the correlation between histopathological grading and the invasion depth of Pi.Results:The 2bRAD-M microbial analysis revealed that the relative abundance of Pi in OSCC tissues (10.80%) was significantly higher than in the HC group (0.50%) ( P=0.001) and OLK group (0.70%) ( P=0.002). FISH probe detection showed that the fluorescence intensity of Pi in human OSCC tissues (125.00±25.13) was higher than in the HC group (11.40±25.49), simple hyperplasia OLK (11.75±23.50), and mild dysplasia OLK (26.83±29.51) groups, with statistically significant differences respectively ( P=0.002, P=0.003, P=0.005). However, there was no significant difference compared to moderate to severe dysplasia OLK (47.40±26.88) ( P=0.210). The fluorescence area of Pi in human OSCC tissues (9 255.00±2 048.00) was significantly larger than in the HC group (18.00±40.25), simple hyperplasia OLK group (27.00±54.00), mild dysplasia (76.00±95.19), and moderate to severe dysplasia (1 628.00±1 265.00) groups, with highly significant differences ( P<0.001). There was a significant correlation between the invasive depth of Pi and the degree of histopathological grading ( P<0.001). In mice, the fluorescence intensity of Pi in OSCC tissues (119.00±8.54) was significantly higher than in the HC group (8.17±20.00) ( P<0.01), but showed no significant difference compared with the OLK group (34.33±28.28) ( P>0.05). The fluorescence area of Pi in mice OSCC tissues (9 971.0±2 807.0) was significantly larger than in the HC group (37.83±92.67) and the OLK group (451.6±780.1) ( P=0.006, P=0.043). There is a significant correlation between the depth of invasion of Pi and the degree of histopathological grading ( P<0.001). Conclusions:This study suggests that Pi in oral mucosal tissue may be a potential biomarker for early detection of OSCC and play an important role in the carcinogenesis process of oral mucosa.