幽门螺旋杆菌感染在喉鳞癌发生发展中的作用及机制

Association between Helicobacter pylori and laryngeal squamous cell carcinoma

目的探讨幽门螺旋杆菌(H.pylori)感染在喉鳞癌(LSCC)发生发展中的作用及可能机制。方法选取2016年3月至2018年2月期间就诊的患者124例,包括71例LSCC患者(LSCC组)和53例良性病变患者(对照组),采集喉黏膜组织标本和血清标本,采用PCR法和ELISA法检测两组组织和血清标本中H.pylori表达;分析H.pylori感染与LSCC患者病理特征间的关系;采用免疫组化法和PCR法检测H.pylori感染与未感染LSCC患者喉黏膜组织中RASSF1A和FHIT表达;采用PCR法和Western blotting法检测H.pylori感染Fa Du细胞株RASSF1A和FHIT表达。结果 PCR检测结果显示,LSCC组与对照组H.pylori阳性表达率分别为71.83%(51/71)、26.42%(14/53),两组比较,P<0.001。PCR检测LSCC患者肿瘤组织和癌旁组织结果显示,6例(11.76%)患者只有肿瘤组织H.pylori呈阳性表达,24例(47.06%)患者只有癌旁组织H.pylori呈阳性表达,21例(41.18%)患者肿瘤组织和癌旁组织H.pylori均呈阳性表达,癌旁组织和肿瘤组织+癌旁组织H.pylori阳性表达率高于仅肿瘤组织H.pylori阳性表达率(P<0.05)。ELISA检测结果显示,LSCC组与对照组H.pylori IgG抗体阳性率分别为78.87%(56/71)、64.15%(34/53),两组比较,P=0.069。单因素Logistic回归分析结果提示,H. pylori感染是喉癌的危险因素(OR=7.09,95%CI 3.11～17.28,P<0.001)。纳入年龄、性别、吸烟史、饮酒史等因素,多因素Logistic回归分析结果提示,H.pylori感染是喉癌发生的独立危险因素(OR=6.94,95%CI 2.87～15.31,P<0.001)。经Mann-whitney秩和检验分析,LSCC患者肿瘤不同发病部位、不同T分型及不同TNM分期H.pylori阳性表达率未见统计学差异(P>0.05)。免疫组化法检测结果显示,H.pylori感染与未感染LSCC患者喉黏膜组织中RASSF1A相对表达量分别为4.46±1.93、8.78±1.82,FHIT相对表达量分别为7.52±1.69、2.86±1. 15,二者比较,P均<0. 05。qRT-PCR法检测结果显示,H. pylori感染与未感染LSCC患者喉黏膜组织中RASSF1AmRNA相对表达量分别为0.74±0.10、0.19±0.06,FHITmRNA相对表达量分别为0.42±0.10、1.16±0.12,二者比较,P均<0.05。不同H.pylori干扰量与Fa Du共培养48 h后,随着细菌干扰量升高,RASSF1A相对表达量升高(P均<0.05),而FHIT相对表达量降低(P均<0.05)。qRT-PCR法检测H.pylori干扰量分别为30∶1、100∶1、300∶1时RASSF1A表达量分别为0.20±0.08、0.43±0.10、1.41±0.08,FHIT表达量分别为1.26±0.22、0.70±0.13、0.18±0.10; Western blotting法检测H.pylori干扰量分别为30∶1、100∶1、300∶1时RASSF1A表达量分别为0.53±0.12、0.59±0.10、1.07±0.14,FHIT表达量分别为0.69±0.11、0.42±0.14、0.28±0.09。结论 H.pylori定植于喉癌患者的喉黏膜组织,是喉癌发生的重要危险因素;其机制可能通过影响抑癌基因RASSF1A和促癌基因FHIT的表达而参与调控喉癌的发生。

Objective To examine the association between Helicobacter pylori and laryngeal squamous cell carcinoma( LSCC).Methods Between March 2016 and February 2018,124 patients were randomly enrolled in this study,including 71 with LSCC and53 with vocal cord polyps and epiglottic cysts. The expression of H.pylori in tissues was detected with polymerase chain reaction( PCR) technique,and serum H.pylori IgG was detected using enzyme-linked immunosorbent assay( ELISA). The expressions of RASSF1 A and FHIT in tissues were detected using immunohistochemistry( IHC) and PCR. The expressions of RASSF1 A and FHIT in FaDu cells under H.pylori co-culture conditions were detected using PCR and western blot tests. Results The H.pylori-positive rates in LSCC and control groups were 71.83%( 51/71) and 26.42%( 14/53)( P<0.001). Six patients( 11.76%) only had H.pylori infection in tumor tissues,24( 47.06%) only had infection in adjacent cancer tissues,and 21( 41.18%) had H.pylori infectionin both tumor and adjacent cancer tissues( P<0.05). Positive rates for H.pylori IgG antibody were 78.87%( 56/71) and 64.15%( 34/53) in LSCC and control groups( P = 0.069). H.pylori infection was a risk factor for LSCC by univariate logistic regression analysis( odds ratio [OR]= 7.09,95% confidence interval [CI]: 3.11-17.28,P<0.001). H.pylori infection was an independent risk factor for LSCC by multivariate logistic regression analysis( OR= 6.94,95% CI: 2.87-15.31,P<0.001). Mann-Whitney tests showed no statistically significant differences in H.pylori infection according to different lesion sites,T stages,and TNM stages( P>0.05). The expressions of RASSF1 A in LSCC tissues with and without H.pylori infection were 4.46±1.93 and 8.78±1.82( P<0.05)using IHC. The expressions of FHIT in LSCC tissues with and without H.pylori infection were 7.52±1.69,2.86±1.15( P<0.05)using IHC. The expressions of RASSF1 A mRNA in LSCC tissues with and without H.pylori infection were 0.74±0.10 and 0.19±0.06( P<0.05) using quantitative real-time( qRT)-PCR. The expressions of FHIT in LSCC tissues with and without H.pylori infection were 0.42±0.10 and 1.16±0.12( P<0.05) using qRT-PCR. The expressions of RASSF1 A mRNA in FaDu cells under H.pylori co-culture conditions( 30∶ 1,100 ∶ 1,300 ∶ 1) were 0.20±0.08,0.43±0.10,and 1.41±0.08,and FHIT mRNA expressions were1.26±0.22,0.70±0.13,and 0.18±0.10 by qRT-PCR. The expressions of RASSF1 A mRNA in FaDu cells under H.pylori co-culture conditions( 30∶ 1,100∶ 1,300∶ 1) were 0.53±0.12,0.59±0.10,and 1.07±0.14,and FHIT mRNA expressions were 0.69±0.11,0.42±0.14,and 0.28±0.09 by Western blotting. Conclusion There is significant evidence that H.pylori is present in the mucosa of laryngeal tissues in LSCC. H.pylori may be an important risk factor for the development of LSCC by regulating RASSF1 A and FHIT expression.