TLR4/NO通路在高危型HPV阳性的子宫颈癌组织和细胞中的表达及意义

摘要

目的 初步探索Toll样受体(TLR)/NO通路在高危型HPV阳性的子宫颈癌组织和细胞中的表达及意义.方法 (1)选取2012年11月1日至2013年5月20日在复旦大学附属妇产科医院就诊的高危型HPV阳性的子宫颈鳞癌患者(宫颈癌组)及经病理检查诊断为正常子宫颈的高危型HPV阳性患者(对照组)各36例,Greiss法检测两组患者宫颈管NO含量;免疫组化SP法检测两组患者子宫颈组织中诱导型一氧化氮合酶(iNOS)蛋白的表达;激光显微切割(LCM)技术获取两组患者子宫颈组织中的鳞状上皮细胞,采用逆转录(RT)-PCR技术检测所获鳞状上皮细胞中TLR/NO通路中关键基因[包括TLR3、TLR4、TLR7、TLR8、TLR9、核因子κB(NF-κB)p65、iNOS]mRNA的表达水平;将宫颈癌组和对照组中表达差异最为显著的TLR(为TLR4)用于后续研究.(2)选用子宫颈癌细胞系CaSki(HPV 16 阳性)、HeLa(HPV 18阳性)和C33a(HPV阴性),采用RT-PCR技术和蛋白印迹法(western blot)分别检测CaSki、HeLa和C33a细胞中TLR4、NF-κBp65、iNOS mRNA和蛋白的表达,采用细胞免疫荧光法检测CaSki、HeLa和C33a细胞中TLR4蛋白的表达.结果 (1)宫颈癌组宫颈管NO含量为(42.92±0.36) μmol/L,对照组为(15.49±0.24) μmol/L,两组比较,差异有统计学意义(P＜0.05).宫颈癌组和对照组子宫颈组织中iNOS蛋白的阳性表达率分别为75％(27/36)、6％ (2/36),两组比较,差异有统计学意义(P＜0.05);iNOS蛋白的表达与子宫颈鳞癌患者年龄、肿瘤大小和临床分期无关(P＞0.05);而与有无淋巴结转移、淋巴血管浸润明显相关(P＜0.05).宫颈癌组子宫颈组织鳞状上皮细胞中TLR3、TLR4、TLR7、TLR8、NF-κBp65、iNOS mRNA的表达水平分别为4.80±0.32、7.41 ±0.39、2.90±0.14、2.02±0.26、3.09±0.16、2.88±0.17,对照组分别为1.21±0.12、1.86±0.21、1.25±0.11、1.22±0.14、0.48±0.15、0.27±0.11,宫颈癌组均高于对照组,分别比较,差异均有统计学意义(P＜0.05),其中宫颈癌组与对照组中表达差异最为显著者为TLR4;而两组间TLR9 mRNA表达水平(分别为0.79±0.05、0.93±0.07)比较,差异则无统计学意义(P＞0.05).(2) RT-PCR技术和蛋白印迹法检测显示,HeLa和CaSki细胞中TLR4、NF-κBp65和iNOS mRNA和蛋白的表达水平均高于C33a细胞,分别比较,差异均有统计学意义(P＜0.05).细胞免疫荧光法检测显示,HeLa、CaSki、C33a细胞中TLR4蛋白表达水平(以积分吸光度表示)分别为3 599±427、2080±456、730±96,两两比较,差异均有统计学意义(P＜0.05).结论 高危型HPV阳性的子宫颈癌组织和细胞中TLR4/NO通路关键基因表达水平增高,推测TLR4/NO通路激活可能参与了高危型HPV感染所致子宫颈癌的形成过程.%Objective To explore the role of toll-like receptor (TLR)itric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection.Methods (1)Study was based on 36 women with nonmalignantcervical tissue as control group and 36 women with squamous cell cervical cancer (SCC),all with hrHPV infection which were assessed by using 14 types hrHPV E6/E7 mRNA real-time PCR kit.The amount of NO was detected by Griess reaction,the expression of inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry (IHC).The mRNA expression of TLR3,TLR4,TLR7,TLR8,TLR9,nuclear factor-κB (NF-κB) p65 and iNOS in control and SCC epithelium which was captured by laser capture microdissection (LCM) were determined.(2)The expressions of TLR4 in CaSki,HeLa and C33a were detected by cell immunofluorescence method.The mRNA and protein expression of TLR/NO pathway transduction molecules including TLR4,NF-κBp65 and iNOS in CaSki,HeLa and C33a cell lines were detected by real-time PCR and western blot.Results (1)The level of NO was much higher in SCC group than that in control group [(42.92±0.36) μmol/L vs (15.49±0.24) μmol/L;P＜0.05].iNOS was detected in 75％ (27 cases) of patients with squamous cervical carcinoma,while only 6％ (2 cases) of normal controls were confirmed with positve result (P＜0.05).TLR/NO pathway maybe activated in SCC,for the mRNA levels of TLR3,TLR4,TLR7,TLR8,NF-κBp65 and iNOS increased significantly when compared to control group (all P＜0.05),and the greatest change in the expression level of TLR in SCC was spotted on TLR4(7.41±0.39 vs 1.86±0.21).(2)The results of immunofluorescence showed that TLR4 was located at plasma membrance of hrHPV positive HeLa and CaSki cells,while the integral optical density of TLR4 in HeLa cells (3 599±427) or CaSki cells (2 080±456) were higher than that in C33a cells (730±96;P＜0.05).The mRNA and protein level of TLR4,NF-κBp65 and iNOS in HeLa and CaSki cells were higher than those of C33a cells (P＜0.05).Conclusion TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection,while the pathway may be involved in cervical tumorigenesis with hrHPV infection.