鼻咽癌发生发展过程中EBER1表达的研究

目的：探讨ＥＢＶ与鼻咽癌发生发展的关系及其病理学意义。方法：应用原位杂交技术检测５０例鼻咽癌石蜡切片组织中ＥＢＥＲ１的表达及分布状况。结果：正常鼻咽粘膜上皮及其单纯性增生、鳞状化生上皮ＥＢＥＲ１表达均阴性,鼻咽癌组织中表达率为９８％（４９／５０）,且在转移灶中不丢失。鼻咽异型增生上皮亦可表达ＥＢＥＲ１阳性信号（１６／２０）,结论：ＥＢＶ与鼻咽癌关系密切,ＥＢＶ可能在鼻咽上皮的恶性转化过程中起重要作用,鼻咽上皮的异型性改变尤其是ＥＢＥＲ１阳性者可认为是癌前病变,随访监测可能有利于早期发现鼻咽癌。     

DNA依赖蛋白激酶在鼻咽癌病变中的表达及意义

[目的]研究DNA依赖蛋白激酶(DNA-PKcs)在鼻咽部正常黏膜、鼻咽炎、鼻咽非典型增生及鼻咽癌中的表达,并探讨DNA-PKcs表达与鼻咽癌临床病理参数间的关系.[方法]应用免疫组化EnVision法检测10例鼻咽部正常黏膜、10例鼻咽炎、21例鼻咽非典型增生和53例鼻咽癌组织中DNA-PKcs蛋白的表达水平,并结合临床病理特征进行分析.[结果]DNA-PKcs蛋白在正常鼻咽黏膜及鼻咽炎组织中呈低表达,在鼻咽非典型增生及鼻咽癌组织中呈高表达,阳性表达率分别为0、20.00％、71.42％和64.15％;其表达与鼻咽癌组织分化类型相关(P=0.023),与性别、年龄、临床分期及淋巴结转移无明显相关性(P均＞0.05).[结论]DNA-PKcs在鼻咽癌病变过程中可能发挥一定的作用.     

HSP70 and mucin 5B: novel protein targets of N,N'-dinitrosopiperazine-induced nasopharyngeal tumorigenesis

N,N '-Dinitrosopiperazine (DNP) induces nasopharyngeal carcinoma (NPC) and shows organ specificity to the nasopharyneal epithelium. To investigate its mechanism, the rat NPC model was inducd using DNP. Rat NPC and normal nasopharyngeal cells were obtained from the NPC model using laser capture. The total proteins from these cell samples were separated with two-dimension polyacrylamide gel electrophoresis techniques, and highly expressed proteins (> five-fold) were analyzed using matrix-assisted laser desorption/ionization time of flight and bioinformatics. The results showed that HSP70 and mucin 5B expression increased not only in rat NPC but also in atypical hyperplasia nasopharyngeal tissues, a precancer stage of NPC. High-expression of heat shock protein 70 (HSP70) and mucin 5B was further supported by western blot analysis. The immunofluorescence and western-blotting studies further showed that DNP induced the expression of HSP70 and mucin 5B in a dosage-dependent manner in normal nasopharyngeal epithelia cells. Our data indicate that DNP triggers over-expression of HSP70 and mucin 5B, and is involved in nasopharyngeal tumorigenesis. HSP70 and mucin 5B may be important targets in nasopharyngeal tumorigenesis induced by DNP. ( Cancer Sci 2009; 100: 216–224)     

鼻咽癌癌变过程中EBERs表达状况的研究

本文采用重组质粒ｐＳＰ６５（带有ＥＢＥＲｓ的基因）体外转录合成的地高辛标记的单链反义ＲＮＡ探针，用原位杂交方法检测处于癌变不同阶段的鼻咽活检组织中ＥＢＥＲｓ的表达状况。ＥＢＥＲｓ检出率为：鼻咽癌（２３／２４），正常鼻咽组织，慢性炎症增生及单纯性增生和化生（０／３０），异型增生和化生（５／８），头颈部其它肿瘤（０／５），鼻咽癌颈淋巴结转移灶（１／１），进一步证明：（１）ＥＢＶ与ＮＰＣ密切相关；（２）     

Promoter hypermethylation of multiple genes in nasopharyngeal carcinoma.

PURPOSE: The methylation profile of nasopharyngeal carcinoma (NPC) has been investigated by a candidate gene approach. EXPERIMENTAL DESIGN: Four NPC cell lines, 4 NPC xenografts, 33 NPC primary tumors, and 6 samples of normal nasopharyngeal epithelium were subjected to methylation-specific PCR for analysis of promoter methylation of eight cancer-related genes. These eight genes were RASSF1A, RARbeta2, DAP-kinase, p16, p15, p14, MGMT, and GSTP1. The correlation between methylation status of these genes and clinical features such as stage, local-regional recurrence, distant metastasis, and survival has been analyzed. RESULTS: The incidence of promoter methylation in NPC samples was 84% for RASSF1A, 80% for RARbeta2, 76% for DAP-kinase, 46% for p16, 17% for p15, 20% for p14, 20% for MGMT, and 3% for GSTP1. No methylation of these genes was detected in the six normal nasopharyngeal epithelium samples. All NPC tumor samples in this study displayed aberrant methylation in at least one of these eight genes. No significant correlation between methylation status of these genes and clinical parameters of the patients was found. CONCLUSIONS: A high frequency of aberrant methylation of the 5' CpG island of the RASSF1A, RARbeta2, DAP-kinase, and p16 genes in the present study was noted. Our findings suggest that methylation of the genes in the critical pathways is common in NPC.     

组织芯片技术检测人鼻咽癌组织及细胞株KIAA1173基因的表达

背景与目的:鼻咽癌(nasopharyngealcarcinoma,NPC)致病的分子机制至今仍不清楚,已有研究表明,染色体3p21～22区域存在与鼻咽癌发生密切相关的抑癌基因。KIAA1173基因是定位于3p22.1的一个新的肿瘤相关基因,其与NPC发病的关系尚未见报道。本研究采用KIAA1173基因特异性原位杂交探针,检测其在NPC组织及细胞株中的表达,探讨KIAA1173基因与NPC发病的关系。方法:克隆KIAA1173基因片段(354bp),并制备cDNA探针;采用组织芯片技术,通过原位杂交检测73例鼻咽部不同组织标本(41例NPC、18例鼻咽非典型增生上皮、14例正常鼻咽粘膜上皮)和6种NPC细胞株(CNE1、CNE2、HNE1、HNE2、6-10B、5-8F)中KIAA1173基因mRNA的表达情况。结果:KIAA1173基因在NPC细胞、非典型增生上皮和正常鼻咽粘膜上皮的阳性率分别为21.9%(9/41)、83.3%(15/18)、92.8%(13/14),而6种NPC细胞株均未见表达;在正常鼻咽粘膜上皮和非典型增生上皮中强阳性率分别是64.3%(9/14)和38.9%(7/18),而NPC中无强阳性,在鼻咽部不同上皮组织中的表达差异有显著性(P<0.001);在38例伴有淋巴细胞浸润的NPC组织中,癌细胞与浸润淋巴细胞之间KIAA1173基因表达有显著性差异(P=0.026),并且呈明显负相关(κ=-0.337,P=0.020)。结论:KIAA1173基因在鼻咽部不同组织中表达不同,正常鼻咽上皮强表达,而NPC细胞中低表达甚至不表达,提示该基因可能参与NPC演变的过程。     

诱导型一氧化氮合酶与β-链接素在鼻咽癌中的表达及其意义

背景与目的：关于诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）与β-链接素（β-catenin,β-cat）在鼻咽癌（nasopharyngeal carcinoma,NPC）中的表达及其与NPC发生、发展、浸润和转移的相关性至今未见报道。本研究拟通过观察iNOS与β-cat在NPC中的表达情况,探讨两者的表达及其联合表达在NPC发生、发展、侵袭和转移过程中的相关性。方法：采用免疫组织化学染色法,观察50例NPC石蜡标本和15例正常鼻咽黏膜标体中iNOS、β-cat的蛋白表达情况。结果：正常鼻咽组织中,iNOS均无表达,而β-cat呈膜表达,胞质无表达;NPC中,iNOS阳性表达定位于细胞质,阳性率为74.0%（37/50）,所有切片中β-cat均可见胞质表达增强而胞膜表达减弱或缺失,与正常组比较,差异均有统计学意义。两者的表达在不同临床分期及有无淋巴结转移之间差异有统计学意义。在不同年龄及性别之间差异无统计学意义。两者的胞质表达强度呈正相关（r=0.394,P=0.005）。结论：iNOS和β-cat在胞质内的高表达均与NPC的发生、发展、浸润和转移相关：两者呈正相关,两者的协同作用可能促进NPC的发生、发展、浸润和转移。     

PTEN与Bcl-2在鼻咽癌中的表达及意义

目的 观察PTEN与Bcl-2在鼻咽癌(NPC)中的表达情况,探讨两者的表达及其联合表达在NPC发生、发展、侵袭和转移过程中的可能作用。方法 采用免疫组织化学染色法,观察50例NPC 石蜡标本和15例正常鼻咽黏膜标本中PTEN与Bcl-2的蛋白表达情况。结果 正常鼻咽组织中,PTEN呈强阳性表达,而Bcl-2的表达仅局限在上皮层的基底细胞层。NPC中,PTEN阳性表达率为22.0%(11/50),Bcl-2为82.0%(41/50),与正常组比较,差异均有统计学意义。两者在NPC的表达强度无明显相关(r=－0.109,P=0.453)。结论 在NPC组织中,PTEN的低表达或表达缺失和Bcl-2强阳性表达可能协同参与了NPC的发生,而在NPC的发展、浸润和转移过程中,两者未能表现出明显的相关性。     

实验性大鼠乳腺增生的细胞核形态计量学研究

 应用图像分析技术对大鼠乳腺轻度增生、增生活跃、不典型增生及正常乳腺组织进行细肥核形态学计量研究，检测4项参数，即细胞核面积、周长、最长径和形态因子。结果显示，轻度增生与正常乳腺间无显著性差异（P＞0．05）；增生活跃组与正常组间、不典型增生组与增生活跃组间均有显著或非常显著的差异（P＜O．05，P＜O．01）。表明随着乳腺增生程度的加重，细胞核增大，核形状变异，将增加乳腺癌的危险性。     

β-链接素、诱导型一氧化氮合酶与环氧合酶-２在鼻咽癌中的表达及临床意义

目的　观察β 链接素（β-ｃａｔ）、诱导型一氧化氮合酶（ｉＮＯＳ）和环氧合酶 ２（ＣＯＸ-２）在鼻咽癌（ＮＰＣ）中的表达情况,探讨三者的表达及其联合表达在ＮＰＣ发生、发展、侵袭和转移过程中的可能作用。方法　采用免疫组织化学染色法观察５０例ＮＰＣ石蜡标本和１５例正常鼻咽组织中β-ｃａｔ、ｉＮＯＳ和ＣＯＸ-２的蛋白表达情况。结果　正常鼻咽组织中ｉＮＯＳ、ＣＯＸ-２未见表达,而β-ｃａｔ胞膜表达。ＮＰＣ中,所有病例β-ｃａｔ胞质均呈阳性表达,与正常组比较,差异有统计学意义（χ^２＝２４.２０７,Ｐ＝０.０００）。β-ｃａｔ胞膜表达明显降低,与正常黏膜组比较,差异有统计学意义（χ^２ ＝２４.２６５,Ｐ＝０.０００）;ｉＮＯＳ表达阳性３７例（χ^２＝２５.３７１,Ｐ＝０.０００）,ＣＯＸ-２表达阳性３３例（χ^２＝２０.１０９,Ｐ＝０.０００）,与正常组比较,差异有统计学意义。胞质内β-ｃａｔ、ｉＮＯＳ、ＣＯＸ-２的表达在不同临床分期及有无淋巴结转移之间差异有统计学意义。三者胞质的表达强度均呈正相关（Ｐ＜０.０１）。结论　胞质内β-ｃａｔ、ｉＮＯＳ和ＣＯＸ-２的过度表达与ＮＰＣ的发生、发展、浸润和转移相关;胞质内β-ｃａｔ、ｉＮＯＳ与ＣＯＸ-２的表达强度呈正相关,能同时针对三者起作用的化疗药物具有更好的抗瘤效应或放疗增敏效应。     

Morphometric study on nasopharyngeal carcinoma (NPC)]

Morphometric parameters of carcinoma cells in 78 NPC and columnar epithelium basal cells in 21 of these 78 NPCs were measured by Quantimet 520 analyser. The results showed that: 1. Prominent enlargement of nuclei was a pathognomonic feature of carcinoma cells, except small spindle carcinoma cells, as compared with the normal basal cells, 2. vesicular nucleus cell carcinoma and small spindle poorly differentiated squamous carcinoma had their own characteristic morphometric parameters as compared with those of the ordinary poorly differentiated squamous carcinoma, and 3. there was no significant morphometric difference between vesicular nucleus cell carcinoma and poorly differentiated adenocarcinoma.     

Met protein expression level correlates with survival in patients with late-stage nasopharyngeal carcinoma.

Met tyrosine kinase, the receptor for HGF/SF, is important in various cellular functions, including proliferation, mitogenesis, formation of branching tubules, angiogenesis, and tumor cell invasion and metastasis. However, the role of Met/HGF signaling pathway in nasopharyngeal carcinoma (NPC) has not been evaluated. In this study, we determined the expression profile and clinical correlation of Met/HGF in 66 cases of advanced NPC and the activation mechanisms of Met receptor in five NPC cell lines. Immunofluorescent staining and quantitative image analysis showed that the Met protein was expressed throughout the tumors and normal nasopharyngeal epithelia. Compared with NPC, the Met expression level was higher in columnar nasopharyngeal epithelium but lower in squamous nasopharyngeal epithelium. The normal interstitial stromal tissue expressed the lowest level of Met protein. HGF was detected mainly in the normal interstitial tissue surrounding the tumor. Met protein expression level was inversely correlated with patients' survival time; the correlation coefficient was -0.319 (P = 0.009). The mean survival time was 118 months in low Met expression group versus 52 months in high expression group (P = 0.0004). The cumulative 5-year survival rate was 77.68% in low expression group versus 38.24% in high expression group. The clinical stage was also significantly more advanced in high Met expression group. In the multivariate analysis, both clinical stage and Met protein expression level were independent prognostic indicators for patient survival. All of the five NPC cell lines tested did not express hgf mRNA but expressed met mRNA, and tyrosine phosphorylation of Met protein was mainly induced by exogenous HGF stimulation in these cells. No mutation was found in the tyrosine kinase and the juxtamembrane domains of Met receptor in the five NPC cell lines tested. These results indicate that: (a) high Met protein expression level correlates with poorer survival in late-stage NPC and serves as an independent prognostic indicator; and (b) the Met receptor in NPC is activated by its paracrine ligand HGF from the interstitial tissues rather than by an autocrine loop or its activating mutation.     

Gli1在鼻咽癌组织中的表达及临床意义

目的:探讨神经胶质瘤相关癌基因1(Gli1)在鼻咽癌组织中的表达及其临床意义.方法:免疫组化方法检测89例鼻咽癌组织及21例正常鼻咽组织中Gli1的表达情况,分析Gli1的表达与与鼻咽癌患者临床病理参数及预后之间的关系.结果:Gli1在鼻咽癌组织的表达水平显著高于正常鼻咽组织(P＜0.01),且Gli1的表达水平与鼻咽癌的T分期、淋巴结转移程度及临床分期显著相关(P＜0.05).此外,Gli1蛋白表达越高,患者的生存时间也明显缩短.结论:Gli1在鼻咽癌组织中呈过表达,可能在鼻咽癌的发生、发展中起重要作用,Gli1有可能成为鼻咽癌治疗的新靶点之一.     

鼻咽癌分子遗传学研究进展

探讨鼻咽癌（nasopharyngeal carcinoma,NPC）发生发展的分子遗传学事件及其变异的NPC临床病理变化的影响。对原发性NPC进行杂合性缺失（loss of hetrozygosity,LOH)和比较基因组杂交（comparative genomic hybridization CGH)分析,观察到NPC发生高频率LOH的染色体主要位于1p、3p、9p、9q、11q、13q、14q、16q和19p,并定位了相应的LOH最小丢失区,并发现特定区域LOH与鼻咽癌临床病理有密切关系;LOH最化值高（FAL值）并伴随病人血清高滴度EBV／EA和EBV／VCA抗体的NPC,多表现为T3＋F4期、进展期TNM／Ⅲ Ⅳ期和远处淋巴结转移。NPC发生遗传物质扩增（gain）的染色体主要位于1q、2q、3q、6p、6q、7q、11.2、8q、11q13、12、15q、17q和20q,表明在这些区域可能存在与NPC发生发展相关的癌基因（oncogenes）活化,且1q,8q,18q的坟增和9p的丢失与晚期NPC有密切关系。正常鼻咽上皮和鼻咽上皮不典型生病变,3p区LOH的检出率分别高达74％和75％,表明3p区缺失是NPC发生过程中极早期分子事件。连锁分析表明HLA基因和细胞色素P4502E1酶基因可能是NPC的遗传易感基因,并定位了新的潜在NPC易感基因位点。应用cDNA微阵列技术,发现细胞周期蛋白、抗凋亡因子、某些癌基因／肿瘤抑制基因、生长促进因子、肿瘤发生生长因子和肿瘤血管生长因子等在NPC发生上调控表达;不同临床分析的NPC与正常鼻咽上皮间均存在差异表达。NPC发生遗传不稳定性（缺失和扩增）是常见的分子事件,遗传变异在NPC的发生、发展过程中起重要作用。通过LOH、CGH、连锁分析和微阵列分析确定NPC特异的分子标记物,能提供用于NPC早期诊断和预后判断的分子标记物,并将使我们建立独立于传统临床分期和分型的新的NPC分子分期的分子分型成为可能。     

Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.     

CSPG4基因在鼻咽癌中的表达分析

  目的  研究硫酸软骨素多糖蛋白4（CSPG4）基因在鼻咽癌及对照组织中的表达。  方法  采用实时荧光定量PCR测定CSPG4基因在4例正常鼻咽上皮和18例鼻咽癌组织（其中Ⅱ期6例,Ⅲ期5例,Ⅳ期7例）中的表达情况;通过构建包含92例对照和187例鼻咽癌的组织芯片,应用免疫组织化学检测CSPG4蛋白的表达和分布。  结果  正常鼻咽上皮中CSPG4基因mRNA表达水平较低,在鼻咽癌中CSPG4表达上调且与临床分期呈正相关,差异具有统计学意义（P=0.017）。免疫组织化学结果表明鼻咽癌中CSPG4蛋白表达水平高于对照组,91.98%的鼻咽癌组织中CSPG4为强阳性表达,而对照组的强阳性率为76.09%（P=0.001）。  结论  CSPG4在鼻咽癌中随着临床进展表达逐渐上调,可能参与了鼻咽癌的发生发展,具体机制及临床应用前景值得进一步深入研究。      

细胞角蛋白基因13在鼻咽癌中的表达研究

目的　探讨细胞角蛋白基因 13(Cytokeratin 13,CK13)在人鼻咽癌 (NPC)中的表达。方法应用免疫组化检测了 40例NPC组织和 8例慢性鼻咽炎 (CINE)组织中CK13蛋白水平的表达 ,同时应用Northern杂交对其中 32例NPC和 8例CINE组织进行了mRNA水平检测 ,并分析了CK13表达与NPC临床特征的关系。结果　在NPC中 ,CK13在蛋白质水平和mRNA水平均较CINE显著降低 (P <0 0 1) ,且CK13表达与NPC颈淋巴结转移有显著相关性 (P <0 0 5 ) ,与肿瘤的T分期无显著相关性(P >0 0 5 )。结论　CK13基因的表达可能与NPC组织分化以及颈淋巴结转移有关。