Prohibitin在放射照射诱导鼻咽癌细胞凋亡中的表达及亚细胞定位的变化

目的:探讨放射照射诱导鼻咽癌细胞凋亡中prohibitin(PHB)的表达及亚细胞定位的改变。方法:体外培养鼻咽癌细胞系CNE-1和CNE-2,应用流式细胞术测定4 Gy X线照射前后CNE-1和CNE-2的凋亡率,Western blot检测照射前后PHB和凋亡相关蛋白Bax与P53表达水平的变化,同时应用免疫荧光染色分析上述蛋白亚细胞定位的改变。结果:X线照射后24 h,CNE-1和CNE-2细胞凋亡率、PHB蛋白表达以及凋亡相关蛋白Bax与P53的表达均较照射前显著增加(P<0.05或P<0.01)。PHB和P53在照射前均主要定位于细胞核,少量分布于胞浆,照射后胞浆荧光强度明显增加,细胞核荧光强度下降,表现为出核转运。Bax在照射前后未发现明显的细胞定位改变。结论:放射照射诱导的鼻咽癌细胞凋亡可能与PHB和P53表达及亚细胞定位改变有关。     

放射诱导鼻咽癌细胞凋亡的研究现状

鼻咽癌（NPC）是我国高发的恶性肿瘤之一,放射治疗是其主要的治疗手段。目前研究表明,放射与细胞凋亡存在密切关系,放射可诱导肿瘤细胞发生凋亡,并加速细胞凋亡过程而使更多的肿瘤细胞死亡,从而使肿瘤发生消退。探讨和研究射线与NPC细胞凋亡的关系,对了解放射诱导NPC细胞死亡的分子机制、放疗技术的改进和放射防护等具有重要的现实意义。     

杂合肽CAMEm诱导鼻咽癌细胞凋亡的作用

目的研究杂合肽(cecropinA-melittinhybridpeptidemutant,CAMEm)对鼻咽肿瘤细胞的诱导调亡作用。方法选取鼻咽癌CNE2细胞株为靶细胞,3T3细胞为正常细胞,以MTT比色法和Hoechest33342染色法,应用流式细胞仪和DNA凝胶电泳观察CAMEm对CNE2细胞的诱导调亡作用,检测杂合肽对CNE2细胞和3T3细胞生长抑制作用的时效和量效关系。结果与对照组相比,当5mg/LCAMEm作用12h时,抑制率升高,P<0·05;125mg/LCAMEm作用12h时,抑制率显著升高,P<0·01。0·5mg/L的CAMEm作用12h时,可以观察到调亡小体的产生;0·5、5、10和25mg/LCAMEm诱导CNE2细胞的调亡率分别达14·60%、17·42%、21·80%和36·10%,诱导调亡作用与剂量和时间呈明显正相关。CAMEm对3T3细胞没有显著影响。结论杂合肽CAMEm对CNE2细胞株具有明显诱导调亡作用,而且对正常细胞没有显著杀伤作用。     

金属硫蛋白在消化管癌组织中的表达及意义

目的 :观察金属硫蛋白 (metalloth ionein ,MT)耐药标志物在消化管癌中的表达。方法 :采用SP免疫组化方法 ,检测了173例消化管癌中MT的表达状况。结果 :MT在消化管癌中的总阳性率为 5 9 5 4%( 10 3 / 173 )。在食管鳞癌、胃腺癌和大肠癌中的阳性率分别为 75 0 0 % ( 2 4/ 3 2 )、5 0 65 % ( 3 9/ 77)和 62 5 0 % ( 4 0 / 64 )。MT表达率与癌组织分化程度和有否淋巴结转移有显著相关性 ,P <0 0 5。结论 :MT在消化管癌中有较高表达 ,检测肿瘤中MT对制定化疗方案有指导意义     

HIF-1α在鼻咽癌组织中表达及意义

目的:探讨缺氧诱导因子-lα(hypoxia-induciblefactor-lalpha,HIF-lα)表达与鼻咽癌(NPC)临床及预后的相关性。方法:对2000年4月~2002年4月收治的经病理明确诊断、完成根治性放疗、随访3年以上的97例NPC放射治疗前活检标本(经病理证实均为低分化鳞癌),应用免疫组织化学染色二步法检测HIF-lα在NPC组织中的表达情况,并分析HIF-lα的表达与NPC分期、颈部淋巴结转移、生存率、远处转移和复发之间的关系。结果:NPC组织中HIF-lα表达率为56.71%。HIF-lα表达与鼻咽癌的NPC分期、颈淋巴结转移、3年生存率、远处转移及复发倾向有密切关系。结论:HIF-lα表达对判断鼻咽癌病变进展、预测颈部淋巴结转移、复发有指导意义,是评估预后的重要指标。     

金属硫蛋白2与基质金属蛋白酶9在炎症相关性结肠癌中的表达及意义

目的:研究金属硫蛋白2(MT2)与基质金属蛋白酶9(MMP9)在小鼠的结肠炎及炎症相关性结肠癌(CAC)中的表达情况及相关性.方法:采用氧化偶氮甲烷试剂(AOM)腹腔注射及葡聚糖硫酸钠试剂(DSS)喂药方法,成功建立小鼠的结肠炎-癌模型.使用免疫组化方法观察MT2与MMP9在小鼠结肠炎-癌模型中的表达情况.应用统计学分析MT2与MMP9在小鼠结肠炎-癌模型中的表达相关性.结果:MT2在小鼠的结肠炎及炎癌组织中的表达明显上调,具有统计学意义(P<0.05);在炎症组与炎癌组,MT2与MMP9的表达均存在显著正相关性,具有统计学意义(r=0.949,P<0.001;r=0.859,P=0.001).结论:MT2与MMP9在小鼠的结肠炎和炎症相关性结肠癌中表达的显著正相关性提示它们可能在结肠炎症促进癌变这一过程起到一定作用,有望成为治疗炎症相关性结肠癌的新靶点.     

新疆鼻咽癌226例临床病理分析

目的研究．LMP1在新疆地区鼻咽癌中的表达及EB病毒与鼻咽癌的关系。方法应用免疫组化法检测100例鼻咽癌中EBVLMP1的表达情况。结果100例鼻咽癌中,LMP1阳性表达为60％（60／100）,35例淋巴结转移病例中,LMP1阳性表达为80％（28／35）。EB病毒LMP1阳性表达与鼻咽癌淋巴结转移显著相关,与年龄、性别、民族及组织分型无明显相关性。结论新疆地区鼻咽癌与EBV感染密切相关,EBVLMP1在鼻咽癌发生、发展中扮演重要角色。     

HOXC6在鼻咽癌组织中的表达及临床意义

目的:探讨鼻咽癌患者组织中同源盒基因C6(HOXC6)的表达水平与临床意义。方法:采用免疫组织化学法检测127例鼻咽癌患者的石蜡组织标本中HOXC6蛋白的表达,分析其表达与患者临床特征及预后的关系。在GEO公共数据库中下载鼻咽癌基因芯片数据集GSE12452和GSE13597,分析HOXC6基因在鼻咽癌和正常鼻咽组织间在转录水平上的表达差异;进一步采用GSEA分析方法探讨HOXC6基因在鼻咽癌中可能的作用机制。结果:HOXC6在鼻咽癌组织中表达上调,其水平表达与鼻咽癌的T分期、N分期和临床分期有显著的相关性(P<0.05),且高表达与鼻咽癌的不良预后显著相关(P<0.05)。COX多因素分析显示:N分期（P=0.022）和HOXC6表达水平（P=0.038）是影响鼻咽癌患者预后的独立危险因素。在基因芯片数据集中,HOXC6 mRNA在鼻咽癌组织中的表达水平明显高于正常鼻咽组织(P<0.05);GSEA分析提示,HOXC6基因高表达样本主要富集了细胞凋亡、TNF-α信号通路、IL6/JAK/STAT3信号通路等基因集,影响鼻咽癌的生物学过程。结论:HOXC6基因的表达与鼻咽癌患者的预后存在密切相关性,可作为其独立预后危险因素。     

鼻咽癌组织中核磷蛋白表达水平与患者疗效的关系

目的：探讨核磷蛋白(nucleophosmin, NPM、B23、NO38或NPM1)在鼻咽癌活检组织中的表达水平与增殖指数和患者个体化治疗后疗效的关系。方法：随机选取中山大学肿瘤防治中心2001年1月1日至2003年12月31日期间收治的157例初诊鼻咽癌患者的治疗前活检蜡块,用免疫组织化学法检测鼻咽癌活检组织中NPM1 和Ki67 的表达,并结合相应临床病理资料,应用统计学方法对结果进行分析。结果：NPM1 的表达水平与Ki67的表达水平呈正相关(r=0.445, P<0.01),与淋巴结分期呈负相关(N0, N1, N2和N3; r=-0.235,P<0.01),但与原发灶分期(T1, T2, T3 和T4; r=0.006, P>0.05) 和临床分期(r=-0.74, P>0.05)无相关性。NPM1表达水平与有淋巴结转移组治疗后淋巴结情况呈负相关(r=-0.196, P<0.05),与无淋巴结转移组治疗后原发灶情况呈负相关(r=-0.349,P<0.05)。NPM1表达水平与预后无明显相关性。结论：NPM1 在鼻咽癌组织中高表达并与Ki67 的表达水平呈正相关,NPM1有促进细胞增殖的作用且可能作为一个评估个体化治疗疗效的评价指标。     

鼻咽癌细胞系CNE-2Z放射敏感性异质性与凋亡的关系

目的评估人鼻咽癌细胞系CNE-2Z放射敏感性异质性与凋亡敏感性的相关性。方法用流式细胞仪、荧光显微镜、电镜、Western-blot检测H5和S1凋亡能力的不同及凋亡相关蛋白表达的差异。结果H5、S1两种细胞的凋亡率均随着照射后时间的延长而增加,但H5比S1高且差异有显著性(P<0.05)。结论人鼻咽癌细胞系CNE-2Z放射敏感性的异质性,与细胞受射线照射后凋亡呈正相关,对射线敏感的亚细胞系凋亡率高。     

Apoptosis induced by low-dose paclitaxel is associated with p53 upregulation in nasopharyngeal carcinoma cells.

Paclitaxel exerts its cytotoxic effect by kinetic suppression of microtubules that block cells in the G2/M phase of the cell cycle and trigger apoptosis. To investigate apoptosis induced by paclitaxel in nasopharyngeal carcinoma (NPC), and its possible molecular mechanism of action, the human NPC cell lines HNE-1 (bearing wild-type p53) and CNE-2 (bearing mutant p53) were treated with different concentrations of paclitaxel. Apoptosis was determined by staining with propidium iodide and also by DNA fragmentation. Protein expression levels of p53, bcl-2 and bcl-xl were examined by Western blotting. Activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase (PARP) were also studied in paclitaxel-induced apoptosis. We showed that paclitaxel inhibited growth and induced apoptosis in both cell lines but that the p53 mutant line (CNE-2) was less sensitive to treatment with low-dose paclitaxel. Caspase-3 activity and cleavage of death substrate PARP were significantly increased in a dose-dependent manner, both in parallel with the induction of apoptosis and growth inhibition of NPC cells. We observed a striking increase of p53 protein levels in NPC cells exposed to 1 and 10 nM paclitaxel but a marked inhibition at 100 nM paclitaxel treatment. An inhibitor of caspase, zVAD.fmk, blocked the apoptotic morphologic changes and DNA fragmentation but did not change the rate of cell death or the protein levels of p53, bcl-2 and bcl-xl. In summary, low-dose paclitaxel inhibited cell growth in NPC cells and induced apoptosis possibly by upregulation of p53. In contrast, cell growth and apoptosis induced by a high dose of the drug occurred in a p53-independent manner, which may directly initiate downstream events of apoptosis.     

鼻咽癌组织中Ki-67表达的阳性单位与癌转移、TNM分期及患者生存时间的关系

目的观察ki67在鼻咽癌组织的表达,从量化角度阐明其在鼻咽癌组织中表达的病理学意义。方法常规石蜡包埋、HE切片确诊,用免疫组化SP法检测ki67在鼻咽癌细胞核中的定位和表达,用Leica Q500MC图像分析系统对其表达强度进行定量分析,并用阳性单位（positive unit,PU）反映其表达强度。结果 ki67在鼻咽癌组织中的阳性表达主要定位在细胞核,ki67阳性细胞为核境界清晰的棕色、棕褐色颗粒沉着细胞。本研究显示ki67表达与鼻咽癌患者有无颈淋巴结转移、治疗方式不具有相关性,经统计学检验ki67与鼻咽癌患者有无远处转移、生存期及临床分期有相关性,数据具有统计学意义。有远处转移的PU值（21.00±5.95）高于无远处转移者（16.82±4.55）,t=-3.069,P=0.004,两者的数据具有统计学意义;我们发现不同生存期患者的表达量均有不同,随着生存期的延长,ki67的阳性表达强度逐渐减弱。生存期3年以下PU值（24.39±4.50）高于生存期3～5年的（17.69±2.61）,生存期3～5年的PU值高于生存期超过5年的（15.10±3.51）,三组之间PU值差异具有统计学意义（F=45.484,P=0.000）。随着临床分期I、II、III、IV期的顺序,鼻咽癌患者的ki67的阳性表达强度逐渐增强,PU值逐渐加大,组间差异具有统计学意义（t=7.271,P=0.002）。说明ki67表达阳性者多出现在NPC的晚期。结论 ki67蛋白表达程度对于判断鼻咽癌患者是否存在远处转移和患者生存时间具有重要价值,表明Ki67可作为判定鼻咽癌的恶性程度和预后的指标之一。     

鼻咽癌组织中Cox-2基因表达的临床研究

目的:探讨环氧化酶-2(Cox-2)基因在鼻咽癌(nasopharyngealcarcinoma,NPC)组织中的表达及其临床意义。方法:采用免疫组织化学技术检测86例NPC组织中Cox-2蛋白表达情况,所有病例随访达5年以上,分析其与临床病理及预后的关系。结果:Cox-2在NPC组织中阳性表达率为73·3%(63/86),与原发灶范围(P<0.05)、颈淋巴结转移(P<0.05)和肿瘤分级均密切相关,P<0·05,且与预后有关,生存期≤5年组Cox-2蛋白表达阳性率(84.4%)显著高于生存期>5年组(61.0%),P<0·05。结论:Cox-2在NPC组织中高表达,可作为判断预后的一种有用指标;Cox-2选择性抑制剂有望成为NPC防治的新靶点。     

Expression of mir-34c-5p and mir-150-5pin nasopharyngeal carcinoma and up-regulated expression after invasion and apoptosis of nasopharyngeal carcinoma cells

Objective MiRNAs are closely related to tumors,and we hypothesized there is specific miR expression in nasopharyngeal carcinoma(NPC).We intended to investigate the expression of mir-34c-5p and mir-150-5p in NPC and to investigate the effects of mir-34c-5p and mir-150-5p on apoptosis and invasion following up-regulated expression in HNE1 NPC cells.Methods MiR-34c-5p and miR-150-5p expression levels in 30 individual cases of NPC and nasopharyngitis were detected with gene chip and qRT-PCR techniques.miR-34c-5p and miR-150-5p were transfected into the NPC cell line HNE1 via liposomes.Their expression levels were detected with qRT-PCR,apoptosis was evaluated by flow cytometry,and invasion ability was assessed via Transwell migration assay.Results MiR-150-5p expression levels in NPC and nasopharyngitis were 0.165±0.092 and 1.062±0.280 respectively,and miR-34c-5p expression levels in NPC and nasopharyngitis were 0.417±0.220 and 1.385±0.739,respectively,which indicated miR-34c-5p and miR-150-5p were weakly expressed in NPC.Apoptosis rates in HNE1 cells transfected by miR-34c-5p and miR-150-5p were increased,by 12.7%and 7.6%,respectively,which were significantly higher compared to blank control(3.9%).The Transwell assay demonstrated that invasive HNE1 cell counts were 32.00±2.00 and 28.33±2.08,respectively,compared to 60.66±8.50 in the blank control(P<0.001).Conclusion MiR-34c-5p and miR-150-5p are lowly expressed in NPC,and their down-regulation may be associated with NPC.     

鼻咽癌组织多药耐药相关蛋白表达及其与临床分期关系的研究

目的:探讨多药耐药相关蛋白(mul-tidrugresistance-associatedprotein,MRP)在鼻咽癌中的表达及与临床分期的关系。方法:对73例鼻咽癌采用免疫组化SP法检测鼻咽癌中MRP的表达,并分析其表达与临床分期的关系。结果:鼻咽癌中有较高水平的MRP表达,阳性表达率为68·49%(50/73)。其中,MRP在Ⅰ~Ⅱ期的阳性表达率为58·33%(7/12),Ⅲ~Ⅳ期的阳性表达率为70·49%(43/61);T1~2期的阳性表达率为61·54%(24/39),T3~4期中阳性表达率为76·47%(26/34);N0~1期的阳性表达率为69·70%(23/33),N2~3期中阳性表达率为67·50%(27/40)。以上表达率经检验差异无统计学意义,P>0·05。结论:MRP基因在鼻咽癌中有较高的表达水平,但其表达与临床分期无关。     

人鼻咽癌细胞株自发性凋亡与放射敏感性的研究

  目的   探讨鼻咽癌细胞自发性凋亡对放射敏感性的预测意义及自发性凋亡水平差异的分子学基础。   方法   克隆形成实验测定人鼻咽高分化鳞癌细胞株(CNE-1)、人鼻咽低分化鳞癌细胞株(CNE-2)接受Varian加速器单野照射(6 MV X线)的存活分数, 单击多靶和线性二次模型拟合辐射剂量存活曲线并求出放射生物学参数; 流式细胞术检测自然生长2、4、6、8d细胞凋亡情况; RT-PCR、Western blot检测凋亡相关基因Bcl-2、Bcl-xl、Bcl-w、Bax、Bad、Bid、Bak转录及蛋白表达水平。   结果   函数模型参数显示CNE-2较CNE-1具有更高的辐射敏感性; 相同培养天数下CNE-2早期及晚期凋亡率显著高于CNE-1(P < 0.05);CNE-2中Bcl-2、Bcl-xl、Bcl-w、Bax、Bad、Bid、Bak基因转录及蛋白表达水平均高于CNE-1(P < 0.05)。   结论   自发性凋亡可能成为放射敏感性的预测指标, 自发性凋亡水平差异可能与凋亡相关基因差异性表达有关。      

不同中医临床证型下行型鼻咽癌组织细胞核蛋白质差异表达的初步研究

目的： 研究下行型鼻咽癌不同中医临床证型患者原发病灶组织细胞核差异表达蛋白质,为中医证型分类的科学性提供依据。方法：采用双向凝胶电泳及基质辅助激光解吸电离-飞行时间质谱法 (MALDI-TOF-MS)对下行型鼻咽癌火毒困结型、气阴两虚型和气血凝结型患者的鼻咽癌组织细胞核蛋白进行分离和表达谱研究,利用生物信息学方法鉴定差异蛋白质。结果：与下行型鼻咽癌气阴两虚型细胞核蛋白点相比,气血凝结型有28个蛋白点表达上调、12个蛋白点表达下调,火毒困结型有9个点表达上调,19个蛋白点表达下调;与下行型鼻咽癌气血凝结型细胞核蛋白点相比,火毒困结型有25个蛋白点表达上调、18个蛋白点表达下调。MALDI-TOF-MS成功鉴定出3个差异蛋白质,来源于气血凝结型鼻咽癌患者的瘤组织细胞不均一性核糖核蛋白H (heterogeneous nuclear ribonucleoprotein H,hnRNP H)和微管蛋白β链1 (tubulin beta-1 chain,TUB1)表达上调,来源于火毒困结型鼻咽癌患者瘤细胞的核蛋白Ⅵ型胶原蛋白α2链 (type Ⅵ collagen alpha 2 chain,COLⅥA2)前体表达下调。结论：下行型气血凝结型鼻咽癌患者的瘤细胞核蛋白hnRNP H和TUB1表达上调,下行型火毒困结型鼻咽癌患者的瘤细胞核蛋白中COLⅥA2前体表达下调。     

三氧化二砷诱导人鼻咽癌细胞凋亡的实验研究

目的 :研究三氧化二砷 (As2 O3 )体外抑制人鼻咽癌细胞株 (CNE1)增殖及诱导凋亡的作用。方法 :以不同浓度的As2 O3 处理CNE1,用四甲基噻唑蓝 (MTT)法测定细胞增殖情况 ,细胞染色方法观察细胞凋亡的形态 ,原位末端标记法 (TUNEL)检测凋亡细胞 ,流式细胞仪检测细胞的DNA含量。结果 :As2 O3 明显抑制人鼻咽癌细胞株CNE1细胞的增殖 ,其抑瘤作用优于顺铂 (DDP)和 5 -氟尿嘧啶 (5 FU)。As2 O3 作用于细胞后 ,可见典型凋亡细胞的形态学改变 :细胞核固缩 ,染色质凝集 ,核碎裂 ,凋亡小体形成等。流式细胞仪DNA线条图上出现典型的亚二倍体“凋亡峰”。结论 :As2 O3 可明显抑制人鼻咽癌细胞株CNE1的生长和促进细胞凋亡的作用     

Potential efficacy of p16 gene therapy for EBV-positive nasopharyngeal carcinoma

The p16 cell cycle inhibitory gene is a potentially critical molecular abnormality in nasopharyngeal carcinoma (NPC). Its expression is silenced through either deletion or promoter methylation in the vast majority of NPC. This in turn is associated with absent or reduced protein expression, which has been previously demonstrated by our group to correlate with inferior clinical outcome. Therefore, we were interested in evaluating the potential of adenoviral mediated p16 gene therapy (adv.p16) in an EBV-positive NPC model (C666-1). We confirm that under basal conditions, p16 protein is undetectable in C666-1 cells, which, in turn, is associated with retention of retinoblastoma protein (pRb) expression. P16 expression was observed as early as 4 hr after infection of C666-1 cells with adv.p16 (10 pfu/cell) with no discernible perturbation in pRb for up to 24 hr. At 48 hr post-infection, p16 expression continued to increase, but at this point, pRb expression started to decline significantly. Cell viability decreased in a dose-dependent manner, down to 20% using 50 pfu/cell of adv.p16. The addition of radiation therapy (RT) administered 24 hr post-infection achieved only a slightly additive cytotoxicity. Adv.p16 therapy resulted in multiple mechanisms of cytotoxicity, including cell cycle arrest at the G0/G1 phase, induction of senescence, along with apoptosis. Ex vivo infection of C666-1 cells with adv.p16 (25 pfu/cell) with subsequent implantation into scid mice completely prevented tumor formation, followed for up to 51 days. Our study demonstrates the potential efficacy of adv.p16 gene therapy for NPC, mediated through multimodal mechanisms of cytotoxicity. Future evaluations will examine strategies to increase in vivo tumor transduction with a view towards future clinical applications.     

Increased expression of SHP-1 is associated with local recurrence after radiotherapy in patients with nasopharyngeal carcinoma

Background

Nasopharyngeal carcinoma (NPC) is a major cancer in southern China. Src homology phosphatase-1 (SHP-1) is a tyrosine phosphatase that regulates growth, differentiation, cell cycle progression, and oncogenesis. We determined the clinical significance of SHP-1 expression in the tumours of NPC patients from southern China who were treated with radiotherapy.

Patients and methods.

SHP-1 expression was determined by real-time polymerase chain reaction (PCR) and western blotting of NPC tissue samples of 50 patients and nasopharyngeal tissues of 50 non-NPC patients who had chronic nasopharyngeal inflammation. SHP-1 expression was measured in NPC tissue samples of 206 patients by immunohistochemistry and survival analysis was performed.

Results

The tumours of NPC patients had significantly increased expression of SHP-1 at mRNA and protein levels relative to patients with chronic nasopharyngeal inflammation. Survival analysis of NPC patients indicated that SHP-1 expression was significantly associated with poor local recurrence-free survival (p = 0.008), but not with nodal recurrence-free survival, distant metastasis-free survival, or overall survival.

Conclusions

SHP-1 appears to be associated with radiation resistance of NPC cells and can be considered as a candidate marker for prognosis and/or therapeutic target in patients with this type of cancer.