RASSF2A基因甲基化在喉癌组织中的表达及意义

目的 探讨抑癌基因RASSF2A表达水平及其基因异常甲基化与喉癌的关系及意义。 方法 采用RT-PCR及甲基化特异性PCR(MSP)检测50例喉癌组织(实验组)和10例癌旁正常黏膜组织(对照组)中RASSF2A基因mRNA的表达水平及其启动子甲基化状态。 结果 喉癌组织中有54%(27/50)存在RASSF2A基因启动子,而对照组正常黏膜组织中未发现RASSF2A基因甲基化现象,差异有统计学意义(χ2=9.818, P=0.002)。RASSF2A甲基化水平程度与喉癌患者临床病理特征无明显相关性。喉癌组织中RASSF2A基因mRNA的表达水平明显低于正常黏膜组织(χ2=9.818, P=0.002)。RASSF2A基因甲基化的喉癌组织中其mRNA的表达减少,反之亦然。 结论 喉癌组织中RASSF2A基因CpG岛甲基化与其基因表达缺失有关,可能参与了喉癌的发生发展过程。     

Inhibitory effects of cucurbitacin B on laryngeal squamous cell carcinoma

Cucurbitacins are compounds isolated from various plant families, which have been used as folk medicines for centuries in countries such as India and China because of their wide spectrum of pharmacological activities such as cytotoxic, anti-inflammatory, and anticancer effects. Accumulated evidences have shown that cucurbitacin B inhibits the growth of numerous human cancer cell lines and tumor xenografts. To determine whether cucurbitacin B can inhibit the growth of laryngeal squamous cell carcinoma, in the present study we investigated the antitumor effect of cucurbitacin B on Hep-2 cells. Hep-2 cells were treated with different concentrations of cucurbitacin B for different time. Cell proliferation, cell cycle distribution, and cell apoptosis were evaluated using MTT assay, flow cytometry, and fluorescent microscopy. It was found that cucurbitacin B exhibited significant efficacy in growth inhibition, cell cycle arrest at G(2)/M phase, and apoptosis induction in a dose- and time-dependent manner. Measuring the modulation of regulators in the cell cycle, apoptosis and signal transductions by Western blot analysis showed that the effect of cucurbitacin B was due to suppression of the expression of p-STAT3, Bcl-2, and cyclin B1. Moreover, in vivo studies were performed in a mouse xenograft model, where cucurbitacin B inhibited tumor growth in a dose-dependent manner. In conclusion, the antitumor effect of cucurbitacin B on Hep-2 cells was due to the induction of cell cycle arrest as well as apoptosis. The possible mechanisms underlying the action might be attributed to the suppression of STAT3 phosphorylation. This investigation suggests a potential clinical application of cucurbitacin B for the treatment of laryngeal cancer patients.     

p53蛋白在喉鳞癌及癌旁组织中表达的研究

应用抗ｐ５３单克隆抗休琢ＬＳＡＢ免疫组织化学染色法检测喉鳞癌，喉癌旁组织及声带息肉ｐ５３蛋白的表达。结果显示：２５例喉鳞癌呈阳性反应，４例距肿瘤边缘〈０．５ｃｍ的喉癌旁组织呈弱阳以应，２０例距肿瘤边缘〉２ｃｍ的喉癌旁组织及１６例声带息肉均为阴性。     

人乳头状瘤病毒感染与国内喉鳞状细胞癌相关性的Meta分析

目的为了综合评价国内喉鳞状细胞癌(LSCC)患者中人乳头状瘤病毒(HPV)阳性表达的流行现状。方法利用计算机全面、系统地检索各数据库,根据纳入及排除标准收集文献,应用Stata 14.0统计学软件对所提取的数据进行Meta分析,对收集的数据总体情况进行分析,并以地域因素、患者性别、HPV分型（高危型和低危型）、吸烟史、饮酒史、癌症临床特征（分化程度、原发部位及癌症分期）进行亚组分析,采用Revman 5.3统计学软件对亚组分析的结果进行统计学检验,最终得到国内喉癌患者HPV感染情况。结果共纳入13篇文献,国内喉癌患者中HPV总阳性率为12％（95％CI：9％～15％）;亚组分析结果：国内喉癌患者中高危型HPV阳性率［10％（95％CI：7％～13％）］较低危型HPV阳性率［3％（95％CI：1％～4％）］高,组间差异具有统计学意义（P<0.00 001）;无吸烟史的喉癌患者HPV阳性率［19％（95％CI：12％～26％）］较有吸烟史喉癌患者HPV阳性率［7％（95％CI：2％～12％）］高,差异具有统计学意义（P=0.004）;无饮酒史的喉癌患者HPV阳性率［12％（95％CI：9％～16％）］较有饮酒史的喉癌患者HPV阳性率［5％（95％CI：3％～8％）］高,差异具有统计学意义（P=0.001）。北方喉癌患者中HPV阳性率［12％（95％CI：7％～17％）］与南方喉癌患者HPV阳性率［13％（95％CI：9％～17％）］、女性喉癌患者HPV阳性率［26％（95％CI：3％～55％）］与男性喉癌患者HPV阳性率［11％（95％CI：7％～15％）］、中低分化的喉癌患者HPV阳性率［15％（95％CI：8％～21％）］与高分化的喉癌患者HPV阳性率［9％（95％CI：6％～13％）］、声门型喉癌的HPV阳性率［10％（95％CI：6％～13％）］与非声门型喉癌患者HPV阳性率［13％（95％CI：7％～19％）］、Ⅰ、Ⅱ期喉癌患者的HPV阳性率［16％（95％CI：10％～21％）］与Ⅲ、Ⅳ期喉癌患者的HPV阳性率［17％（95％CI：10％～25％）］对亚组间差异的检验结果显示无明显统计学意义。结论本研究通过对全国喉癌患者HPV阳性率进行Meta分析,进一步明确了HPV与喉癌患者基本特征之间的关系。国内喉癌患者中存在较低的HPV阳性表达,其中高危型HPV阳性率较低危型高,无吸烟史或饮酒史的喉癌患者HPV阳性率更高。此研究结果对了解国内喉癌发生危险因素的分析有一定的参考价值,可用来指导HPV阳性喉癌患者的个体化治疗方案。     

幽门螺旋杆菌感染在喉鳞癌发生发展中的作用及机制

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FaDu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.pyloriIgG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FaDu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。     

Retrospective analysis of 659 laryngeal squamous cell carcinoma patients treated with open laryngeal function-preserving operations

Abstract

Background: Open laryngeal function-preserving surgery is a common therapeutic strategy for Chinese laryngeal squamous cell carcinoma (LSCC) patients.

Objectives: To explore the fundamental clinical characteristics and survival prognostic factors of LSCC patients treated with open laryngeal function-preserving operations.

Material and methods: about a total of 659 LSCC patients undergoing open laryngeal function-preserving operations were retrospectively reviewed. Kaplan–Meier method, Log-rank test and Cox regression model were performed in survival analyses.

Results: About 612 patients were glottic cancer, followed by 44 cases in supraglottis and 3 cases in subglottis. The number of patients in stage T1, T2, T3 and T4 was 190, 365, 100 and 4, respectively. Nineteen patients had positive cervical lymph nodes. The 5-year overall survival, disease-free survival and disease-specific survival rates were 90.1, 84.9 and 90.7%, respectively. T stage and second primary tumour were independent factors predicting overall survival, while T stage and pathological differentiation were independent risk factors for tumour recurrence and progression.

Conclusions and significance: Open laryngeal function-preserving surgery is a crucial therapeutic strategy for Chinese patients with early and, in some cases, advanced-stage LSCC. Early diagnosis and therapy and sufficient and safe surgical margins are key to improving the survival rate and reducing the risk of relapse in LSCC patients.     

线粒体基因组单倍型类群与喉鳞状细胞癌的相关性研究

目的检测常见线粒体基因组（mtDNA）单倍型A、B、C、D、F、G、M在正常人群及喉癌患者中的分布频率,探讨mtDNA单倍型与喉鳞状细胞癌（laryngeal squamous cell carcinoma, LSCC）发病的相关性。方法选取2013年12月~2015年12月80例LSCC患者作为实验组,同期选取健康体检者74例作为对照组（normal control, NC）,提取两组病例的全血DNA,经PCR-扩增、基因测序,划分单倍型及其子单倍型,计算mtDNA单倍型及其子单倍型在不同组的分布频率,运用SPSS 20.0统计学软件进行统计分析,P<0.05为有统计学意义。结果mtDNA单倍型B及D4在LSCC组的分布频率高于NC组(P﹤0.05),且与临床分期无关（P>0.05）。结论mtDNA单倍型B和D4可能是LSCC的风险因素,可以作为LSCC发病的基因筛查参考指标。     

MicroRNA-107抑制喉癌细胞的迁移及侵袭能力的临床分析

目的通过观察microRNA 107（miR 107）在喉癌及癌旁组织中的表达,并且在喉癌细胞系选择性上调及敲减miR 107,检测其对喉癌细胞迁移及侵袭能力的影响。方法收集40例喉癌及其邻近癌旁组织标本,运用RT qPCR检测miR 107的表达,并分析其在喉癌中的临床意义。在人喉癌细胞TU212及TU686中过表达或敲减miR 107,通过Transwell法检测其对喉癌细胞迁移及侵袭能力的影响。结果MiR 107在喉癌组织中的表达显著低于癌旁组织,差异具有统计学意义（P<0.05）,miR 107的表达水平与肿瘤细胞分化程度、淋巴结转移及原发部位相关（P<0.05）。细胞实验显示,过表达miR 107后喉癌细胞迁移及侵袭能力明显下降（P<0.05）,而敲减miR 107后细胞迁移及侵袭能力明显增强（P<0.05）。结论MiR 107在喉癌中表达显著下调,它能够抑制喉癌细胞的迁移和侵袭能力。     

术前外周血纤维蛋白原水平对喉癌预后的影响

目的探讨术前外周血纤维蛋白原(FIB)水平对喉鳞状细胞癌患者预后的影响。方法回顾性分析南京医科大学第一附属医院458例行初次手术治疗的喉鳞状细胞癌患者的临床资料。根据患者术前的FIB,绘制诊断总体生存期(OS)的受试者工作特征曲线(ROC),确定最佳截断值,从而分为低危组和高危组;并采用卡方检验、Kaplan-Meier单因素分析、Cox风险比例模型多因素分析FIB以及其他临床病理参数与喉癌患者总体生存期的关系。结果 FIB诊断喉癌患者OS的ROC曲线下面积(AUC)为0.67。较高纤维蛋白原水平(>3.12 g/L)与肿瘤分期、甲状软骨板侵袭有明显统计学意义(P<0.001)。单因素分析显示FIB、分型、分期、病理分化程度、甲状软骨板累及、淋巴结转移、年龄与喉鳞状细胞癌患者的OS有关;多因素分析显示FIB、分期与年龄是影响喉鳞状细胞癌患者死亡的独立危险因素。结论术前外周血高FIB水平的喉鳞状细胞癌患者术后OS较短,该指标可作为评估喉癌患者预后的重要指标。     

Level IIb lymph node metastasis in laryngeal squamous cell carcinoma

OBJECTIVES: Selective neck dissection, despite preservation of the spinal accessory nerve, can lead to some degree of postoperative shoulder dysfunction as a result of removal of level IIb lymph nodes. The aim of this study was to determine whether level IIb lymph nodes can be preserved in elective or therapeutic neck dissection as a treatment for patients with laryngeal squamous cell carcinoma (SCC). STUDY DESIGN: This was a prospective analysis of a case series. METHODS: A prospective analysis of 65 patients with laryngeal SCC who underwent surgical treatment of the primary lesion with simultaneous neck dissection from January 1999 to December 2002 was performed. During the neck dissection, the contents of the level IIb lymph nodes were dissected, labeled, and processed separately from the remainder of level II nodes and the main neck dissection specimen. The incidence of pathologic metastasis to level IIb lymph nodes and the regional recurrence within this area were evaluated. In addition, several potential risk factors for metastatic disease in the level IIb lymph nodes such as sex, age, cT stage, cN stage, and the presence of other positive lymph nodes were also evaluated. RESULTS: A total of 125 neck dissections were performed in this series. Of these dissections, 102 (82%) were elective and 23 (18%) were therapeutic. The prevalence of metastases in the level IIb lymph nodes was 1% (one of 46) and 0% (zero of 56) in clinically node-negative (N0) ipsilateral and contralateral necks, respectively, and 37% (seven of 19) and 0% (zero of four) in clinically node-positive ipsilateral and contralateral necks, respectively. There was a statistically significant association between level IIb metastases and clinically positive N stage (P<.001). The presence of other positive lymph nodes was also shown to have a statistically significant association with metastasis in the level IIb lymph nodes (P=.001). Only two of 46 patients (4%) with clinically N0 necks developed a regional recurrence. However, three of eight cases (38%) with positive pathologic level IIb lymph nodes developed regional recurrence. CONCLUSION: Level IIb lymph node pads may be preserved in elective neck dissection in patients with laryngeal SCC. However, this area should be removed thoroughly during therapeutic neck dissection in the treatment of clinically node-positive necks.     

垂体肿瘤转移基因在喉鳞状细胞癌中的表达及临床意义

目的探讨喉鳞状细胞癌中垂体肿瘤转移基因(pituitary tumor transforming gene,PTTG)蛋白表达及其临床意义。方法采用免疫组织化学S-P法检测56例喉鳞癌及13例喉正常黏膜中PTTG的表达,分析PTTG表达与喉鳞癌临床病理指标之间的关系。结果 PTTG蛋白在喉鳞癌组织中的阳性表达率为64.3%,在喉正常黏膜细胞中的阳性表达率为23.1%(P<0.05);PTTG的阳性表达及其强度与喉鳞癌临床病理指标之间差异具有统计学意义(P<0.05)。结论 PTTG基因蛋白阳性表达及其强度可做为判定喉鳞癌生物学行为及其预后的参考指标之一。     

Prognostic significance of Ki-67 expression for patients with laryngeal squamous cell carcinoma primarily treated by total laryngectomy

The aim of this study was to determine the prognostic value of Ki-67 immunostaining in laryngeal squamous cell carcinomas. Ki-67 labeling was quantified in 63 laryngeal squamous cell carcinomas by counting at least 1,000 tumor cells in the most immunoreactive area in each sample, and the Ki-67 labeling index was calculated as a percentage. The antigen expression was compared with clinical factors, histopathological grading and prognosis. The Ki-67 mean proliferation index for all patients was 25.44% ( range, 2–75%). A significant correlation was found between Ki-67 mean proliferation index and patient age (P<0.05), T-stage (P<0.05), nodal metastasis (P=0.001) and recurrence (P<0.001). There was no significant association between the Ki-67 mean proliferation index and tumor site or histologic grade. A univariate analysis showed that the Ki-67 labeling index >21% (P<0.001), T-stage (P<0.001) and nodal metastasis (P=0.001) are determinants of recurrence. In the multivariate analysis, the Ki-67 labeling index >21% (P<0.001), T-stage (P<0.001) and nodal metastasis (P<0.05) were independent predictors of recurrence. Kaplan-Meier plots of survival in patients with Ki-67 values above and below the median (21%) of the general study population showed that a high Ki-67 labeling index correlated with a shorter disease-free survival (P<0.0001). The analysis of the Ki-67 labeling index at the time of initial surgery may be a powerful prognostic marker for patients with laryngeal squamous cell carcinoma and may be useful for selecting subgroups of patients who should be treated with more aggressive therapies.     

金丝桃素联合放射线照射对喉癌细胞Hep-2的影响

目的：探讨金丝桃素（HY）联合放射线照射对喉癌细胞Hep-2的影响。方法：体外培养的喉癌细胞Hep-2分别给予0、1、2、3Mg／ml终浓度的HY,加药1h后,给予5Gy的放射线照射（HY加放射组）;另取喉癌细胞Hep-2分别给予5、10、20、25μg／ml终浓度的HY,不给予放射线照射（单纯HY组）;各实验组均设空白对照。继续培养48h后,应用显微镜、MTT法和流式细胞仪（FCM）分析HY加放射组和单纯HY组对喉癌细胞Hep-2的影响。结果：光镜下可见喉癌细胞生长密集,细胞呈梭型或多角型,细胞彼此之间相接触,放射后的喉癌细胞主要改变为部分细胞肿胀,HY和喉癌细胞共同经放射线作用后,在HY低剂量时即可见细胞数量明显减少,肿胀细胞比例明显增多,高剂量时可见细胞坏死。MTT结果显示放射后HY可显著抑制喉癌细胞的生长;FCM分析表明放射后HY可将喉癌细胞阻滞在G0／C1期,并诱导喉癌细胞凋亡。HY单独应用,对喉癌细胞的抑制作用相对较弱。结论：HY联合放射线照射可有效地抑制喉癌细胞的增殖,并可诱导喉癌细胞凋亡。     

Molecular and immunologic analysis of laryngeal squamous cell carcinoma in smokers and non-smokers

Background

Laryngeal squamous cell carcinoma (LSCC) is strongly associated with tobacco use, but recent reports suggest an increasing incidence of LSCC in patients without traditional risk factors, suggesting an alternative etiology of tumorigenesis. The purpose of this study is to characterize this non-smoking population and to compare immunohistochemical markers in tumor specimens from non-smokers and smokers with LSCC.

喉鳞癌组织中浸润淋巴细胞超微结构研究

对１４例喉鳞状细胞癌组织中淋巴细胞（ＴＩＬ）浸润状况及其与癌细胞间的相互关系进行透射电镜观察。结果显示喉鳞癌组织中普通存在着程度不等的淋巴细胞浸润，但多数表现为静止状态，少部分具有活化的母细胞特征且与癌细胞密切接触，使其自身和癌细胞均表现出明显的形态学变化，表明其可探讨ＴＩＬ对肿瘤细胞生长及杀伤机理提供重要的形态学依据。     

端粒酶催化亚单位在喉鳞癌组织中的表达及意义

目的研究喉鳞癌组织中端粒酶催化亚单位（hTERT）的表达及其与肿瘤临床病理参数的关系.方法应用免疫组化法检测39例喉鳞癌组织,10例声带息肉组织中hTERT的表达,并分析喉鳞癌组织中hTERT蛋白的表达与肿瘤各项临床病理参数的关系.结果39例喉鳞癌组织中hTERT蛋白阳性表达率分别为84.6%（33/39）.10例声带息肉组织中hTERT蛋白均未见阳性表达.hTERT蛋白在喉鳞癌组织与声带息肉组织中的表达差异有统计学意义.喉鳞癌组织中hTERT蛋白表达与癌组织的的组织学分级、临床分期、淋巴结转移间均未见相关性.结论hTERT的激活在喉鳞癌的发生中起重要作用.     

CPS1在喉鳞癌组织中的表达及临床意义

目的 研究氨基甲酰磷酸合成酶1(CPS1)在喉鳞癌患者组织中的表达水平及与临床病理指标的相关性,探讨CPS1对喉鳞癌的临床意义。 方法 采用逆转录实时定量聚合酶链反应(RT-qPCR)及Western blot免疫印迹技术,分别检测喉鳞癌组织及癌旁组织中CPS1 mRNA及蛋白表达水平,同时采用免疫组织化学法检测 CPS1在喉鳞癌组织中的表达水平及其结构中的分布情况。 结果 RT-qPCR 和Western blot结果显示,与癌旁组织相比,喉鳞癌组织CPS1 mRNA 和蛋白表达明显增高,表达差异有统计学意义(P<0.01);免疫组化结果显示CPS1表达与肿瘤分化程度、临床分期、淋巴结转移等因素密切相关(P<0.05),而与患者年龄、性别、吸烟史、饮酒史及肿瘤部位无关(P>0.05)。 结论 在喉鳞癌组织中CPS1的mRNA和蛋白表达明显升高,且与临床进展密切相关,说明CPS1在喉癌发生及转移过程中起到一定的作用,可能作为喉癌诊断的重要指标。