不同来源潜伏膜蛋白1基因转染对鼻咽癌CNE1细胞生长的影响

目的:探讨不同来源的EB病毒潜伏膜蛋白1(LMP1)基因转染对人高分化鼻咽癌(NPC)细胞系CNE1生长的影响。方法:采用脂质体介导法分别将含有来源于B95-8淋巴细胞标准株的LMP1基因(B95-8-LMP1)和来源于NPC组织的LMP1基因(CAO-LMP1)的质粒转染CNE1;RT-PCR和蛋白印迹法分别鉴定LMP1mRNA和蛋白表达;结晶紫法、流式细胞术和平板克隆形成法测定不同来源的LMP1对CNE1生长特性的影响。结果:成功建立稳定表达不同来源LMP1的CNE1细胞系。两种不同来源的LMP1均可促进CNE1细胞的生长(P<0.01);CAO-LMP1比B95-8-LMP1具有更强促进CNE1生长的作用(P<0.01)。结论:不同来源LMP1对CNE1的体外生长均有促进作用,CAO-LMP1比B95-8-LMP1具有更强的促增殖能力。     

过表达EB病毒潜伏期膜蛋白1(LMP1)的人B淋巴细胞株的建立

目的构建EB病毒潜伏期膜蛋白1(LMP1)基因的真核表达载体,通过电穿孔法转染人B淋巴瘤细胞株Ramos细胞,建立稳定的LMP1表达细胞株。方法扩增带有EcoRⅠ与BamHⅠ酶切位点的LMP1编码基因,克隆至pcDNA3.1-EF1a-mcs-3FLAG-CMV-EGFP真核表达载体,挑取阳性单克隆感受态细胞行PCR以及测序后将重组质粒通过电穿孔法转染Ramos细胞, G418筛选稳定表达LMP1的细胞株, PCR和Western blot法分别检测LMP1 mRNA和蛋白在Ramos细胞中的表达。结果 PCR与测序证实成功构建了LMP1重组质粒,建立了稳定转染的Ramos细胞,且LMP1蛋白可在细胞中成功表达。结论成功建立了稳定表达LMP1的Ramos细胞。     

EB病毒LMP1表达对鼻咽癌细胞增殖及细胞周期分布的影响

目的:观察EB病毒潜伏膜蛋白1(LMP1)表达对鼻咽癌细胞系增殖和细胞周期分布的作用。方法:用免疫组化(LSAB)法检测LMP1蛋白的表达;用MTT法测定鼻咽癌细胞系的增殖能力;用流式细胞术检测细胞周期的分布。结果:表达LMP1的鼻咽癌细胞系L-CNE1的OD值明显高于LMP1阴性的鼻咽癌细胞系V-CNE1和CNE1(P＜0.001)。L-CNE1细胞系的G₁期细胞百分率明显减少和S期细胞百分率明显增加,与V-CNE1和CNE1细胞系比较均有非常显著性差异(P＜0.001)。而V-CNE1与CNE1细胞系之间各期细胞百分率则无明显差异(P＞0.05)。结论:EBV-LMP1表达可使鼻咽癌细胞系细胞周期分布改变和增殖能力增强。     

不同辐射抗拒鼻咽癌细胞微小RNA差异表达的研究

目的: 在验证鼻咽癌（nasopharygycarcinoma, NPC）细胞CNE-1和CNE-2不同辐射抗拒性的基础上，探索微小RNA(miRNA)在CNE-1和CNE-2中的表达差异。方法: 通过观察X线照射对CNE-1和 CNE-2 细胞克隆形成数目的影响，利用SigmaPlot 软件进行分析及线性二次模型拟合存活曲线, 比较CNE-1和CNE-2 细胞剂量存活曲线及其生物学参数；采用Paraflo microfluidic microRNA芯片检测，用激光扫描器收集杂交的图像，经LOWESS 滤器规范信号后分析数据差异,根据Targetscan3.1数据库资料 (http:∥www.targetscan.org)，预测CNE-1和CNE-2细胞microRNA差异表达与NPC放射敏感性差异的关系。结果: 发现在CNE-1和CNE-2细胞中miRNA的差异表达，即与CNE-2细胞比，在检测的326个microRNA中，CNE-1细胞中有20个miRNA上调，13个miRNA下调，其中检测量的绝对值在2 000以上且两者相差在3倍以上的miRNA有hsa-miR-152、hsa-miR-7、hsa-miR-205和hsa-miR-572；分析结果提示明显差异表达的miRNA与放疗敏感性密切相关。结论: 不同辐射抗拒NPC细胞株CNE-1和CNE-2细胞的microRNA的表达有差异；差异表达的miRNA与放疗敏感有关。     

鼻咽癌组织中EB病毒潜伏相关基因的表达

目的： 通过检测鼻咽癌组织中EB病毒的潜伏膜蛋白LMP1的序列以及LMP1、EBNA1、EBNA2的mRNA表达来探讨EB病毒的感染状态及其表达产物与鼻咽癌的关系。方法: 应用PCR法检测鼻咽癌组织中LMP1 DNA的存在，并对鼻咽癌来源的LMP1和EB病毒永生化狨猴B淋巴细胞系B95-8来源的LMP1进行测序，比较序列的差异。利用巢式RT-PCR检测鼻咽癌组织中LMP1、EBNA1、EBNA2的mRNA表达。结果: 47例鼻咽癌组织均含有LMP1 DNA，所有鼻咽癌来源的LMP1 DNA与B95-8来源的LMP1 DNA序列比较均存在着多个单核苷酸变异，最明显的是XhoⅠ酶切位点的丢失。测序后显示鼻咽癌来源的LMP1 DNA有30个核苷酸的丢失。巢式RT-PCR显示LMP1、EBNA1、EBNA2在鼻咽癌中的mRNA表达率分别为76.6％、80.0％和74.5％。其中EBNA1的表达是由Qp启动的，而B95-8细胞中EBNA1的表达是由Cp启动的。结论: 鼻咽癌中EB病毒的作用途径比较复杂，LMP1、EBNA1、EBNA2等潜伏期基因还有早期裂解基因BARF1均可能参与鼻咽癌的发生发展过程。     

HMG-CoA2裂解酶在鼻咽癌组织中表达及其对鼻咽癌细胞增殖、侵袭能力的影响

目的探讨HMG-CoA2裂解酶(HMGCL)在鼻咽癌(NPC)组织中表达及其过表达对鼻咽癌细胞行为学的影响。方法 Western blot检测64例鼻咽癌组织和42例正常鼻咽上皮组织(NNE)及5个NPC细胞系(HK-1、CNE1、CNE2、HONE1和HNE-1)和正常鼻咽上皮细胞NP69中HMGCL表达水平;以HK-1细胞为研究对象,构建过表达HMGCL的细胞株;MTT法、集落形成实验、Transwell实验分别检测过表达HMGCL后HK-1细胞增殖、集落形成和迁移侵袭能力。结果 HMGCL在NPC和NPC细胞系中普遍低表达(P0.05),在NNE和NP69细胞中普遍高表达(P0.05);成功构建了过表达HMGCL的HK-1细胞株,过表达HMGCL后HK-1细胞增殖、集落形成和迁移侵袭能力均显著降低(P0.05)。结论 HMGCL在鼻咽癌组织中低表达可能与鼻咽癌细胞恶性表型有关,为将来寻找抑制鼻咽癌发生进展提供了新的靶标。     

EB病毒潜伏膜蛋白1对鼻咽癌细胞增殖及STAT3信号通路的影响

目的:探讨EB病毒编码的潜伏膜蛋白1(LMP1)对人鼻咽癌细胞增殖及信号转导与转录活化因子3(STAT3)信号通路的影响.方法:体外培养人高分化鼻咽癌细胞系CNE1及转染了LMP1基因质粒的CNE1细胞(CNE1-GL细胞),通过绘制细胞生长曲线及平板克隆形成实验比较转染LMP1基因前后细胞增殖能力的变化;RT-PCR及免疫印迹方法检测STAT3及其下游基因survivin表达的变化.结果:转染LMP1基因后,CNE1细胞生长加快、克隆形成率增加;STAT3活化程度增加,survivin表达增强.结论:LMP1可促进CNE1细胞增殖,其机制可能是通过活化STAT3信号通路,进而促进抗凋亡基因survivin表达.     

LMP1-CTAR3对鼻咽癌干细胞SP18生长的影响

背景：课题组前期构建了CTAR3缺失突变型LMP1(LMP1△232-351)表达载体,初步了解该活性区在LMP1发挥作用中扮演十分重要的角色。 目的：观察LMP1-CTAR3对鼻咽癌干细胞SP18生长的影响。 方法：采用逆病毒感染方式,建立稳定表达LMP1及CTAR3缺失突变型LMP1(LMP1△232-351)的鼻咽癌干细胞SP18细胞系,通过细胞生长曲线、软琼脂集落形成实验和平皿克隆形成实验观察LMP1△232-351对鼻咽癌干细胞SP18生长的影响,然后选用流式细胞术检测细胞周期,计算细胞增殖指数。另外,利用Western-blot检测细胞中JAK3蛋白的磷酸化水平。 结果与结论：①与SP18-LMP1细胞比较,SP18-LMP1△232-351细胞生长速度减慢,增殖指数降低(P < 0.01)。②在SP18-  LMP1△232-351细胞中JAK3的磷酸化水平低于SP18- LMP1细胞。结果说明LMP1-CTAR3可能通过影响SP18细胞中JAK3的磷酸化,下调细胞的增殖指数,降低促细胞增殖的能力。     

前梯度蛋白2干扰质粒的构建及其对鼻咽癌细胞生物学功能的影响

目的:构建前梯度蛋白2(AGR2)基因干扰重组质粒及建立AGR2基因沉默的鼻咽癌细胞及动物模型,检测AGR2对鼻咽癌细胞的生物学功能的影响。方法:首先以real-time PCR和Western blot验证AGR2在鼻咽癌细胞系5-8F和6-10B中的表达,以高表达AGR2的5-8F细胞作为干扰转染的目标细胞;设计及构建shRNA表达载体pSR-GFP/Neo-AGR2-shRNA,将其转染至5-8F鼻咽癌细胞系中,通过Transwell小室迁移及侵袭实验来检测其迁移和侵袭功能的变化。结果:与6-10B细胞比较,AGR2在5-8F中的表达显著上调(P0.05);干扰鼻咽癌细胞5-8F中的AGR2可使细胞迁移和侵袭功能明显减弱(P0.05),使其体外成瘤能力显著降低(P0.05)。结论:AGR2在鼻咽癌细胞系5-8F中明显高表达;pSR-GFP/Neo-AGR2-shRNA可成功干扰5-8F中AGR2的表达;AGR2可抑制5-8F细胞的迁移、侵袭及体内成瘤能力。     

潜伏期膜蛋白1与疾病关系的研究进展

EB病毒(EBV)是一种普遍存在的致癌病毒,是高度相关的淋巴和上皮肿瘤的来源和发展,包括伯基特淋巴瘤和鼻咽癌(NPC)等,EBV基因几乎可以在所有细胞中。EBV感染通常与少数潜伏病毒功能的蛋白质表达,包括潜伏膜蛋白LMP1和LMP2A等和巴尔核抗原1(EBNA1)。LMP1是肿瘤坏死因子受体超家族的成员,被认为是EBV的主要致瘤蛋白。在EB病毒中通过2个C末端结构域编码基因蛋白LMP1信号来驱动细胞生长,存活和转化。LMP1蛋白目前是惟一已被证实的EB病毒的癌基因,LMP1的表达参与了肿瘤的发生与发展,是目前癌症方面研究的重点。     

巨噬细胞移动抑制因子提高鼻咽癌细胞体外侵袭能力

目的　研究巨噬细胞移动抑制因子 (MIF)体外提高鼻咽癌细胞侵袭能力的原因 ,探讨鼻咽癌细胞早期发生侵袭和转移的机制。方法　采用微孔迁移分析法 (micron migrationassay)检测鼻咽癌细胞株CNE 1、CNE 2在细胞因子MIF诱导下通过 8μm直径微孔的细胞数量变化 ,采用Western蛋白印迹法、流式细胞术和酶联免疫吸附法检测侵袭转移相关因子基质金属蛋白酶 2、9(MMP2 ,MMP9)及白介素 8(IL 8)在此过程中的相应改变。结果　 (1)经MIF诱导后 ,CNE 1通过 8μm微孔的细胞数由原来的 2 3 7± 11 0 2增加至 113 7± 2 0 9,CNE 2则由原来的 3 4 7± 7 41增加至 3 11 3± 48 9,差异均呈显著性 (P =0 0 0 5,P =0 0 0 1)。 (2 )经MIF诱导后 ,癌细胞表达MMP9蛋白的细胞比例均明显增加 ,CNE 1细胞由 2 8 5%± 2 45%增加至 82 4%± 3 49% (P =0 0 0 1) ,而CNE 2细胞则由刺激前的 3 2 8%± 3 48%增加至 86 1%± 1 62 % (P =0 0 0 2 )。同时癌细胞MMP9蛋白表达强度也显著增强 ,CNE 1和CNE 2细胞MMP9蛋白的平均相对强度均比诱导前提高 3倍以上 (P <0 0 5)。但MIF刺激前后 ,MMP2蛋白的细胞表达数量和表达强度在两株细胞中却未见明显变化 (P值均大于 0 0 5)。 (3 )MIF诱导后 ,CNE 2细胞培养上清液中的IL 8浓度为 12     

Detection of Epstein-Barr Virus DNA in well and pooly differentiated nasopharyngeal carcinoma cell lines

Undifferentiated and poorly differentiated nasopharyngeal carcinoma (NPC) were know to be tightly associated with Epstein-Barr Virus (EBV). Its association with well differentiated NPC was also reported. In the present study, the presence of EBV was investigated by nucleic acid hybridization, Polymerase Chain Reaction (PCR), Immunoblot andin situ hybridization in two well differentiated NPC cell lines (CNE-1 and HK-1) and two other poorly differentiated NPC cell line (CNE-2 and CNE-3). Contrary to previous report indicating the absence of EBV in these cell lines, EBV DNA and proteins were present in all cell lines. The detection of EBV became more easily when the investigation was carried out on the nude mice tumor induced by transplantation of each NPC epithelial cell line. The EBV latent membrane protein (LMP1) was found byin situ hybridization to be intergrated partly in the chromosomal DNA of these cell lines. The observations indicate that EBV could persist for a long time in the carcinoma cells established directly from well and poorly differentiated tumor biopsies and from transplantable NPC tumor in nude mice.     

Upregulation of LMP1 Expression by Histone Deacetylase Inhibitors in an EBV Carrying NPC Cell Line

OBJECTIVES: In about 60% of Epstein-Barr virus (EBV) carrying nasopharyngeal carcinomas (NPC) LMP1 expressing cells can be detected. The frequency of LMP1 positive cells and the expression level varies from cell to cell in the different tumors. Cell lines derived from EBV positive NPCs loose the virus during in vitro culture. The in vitro infected NPC cell line TWO3-EBV used in our study carries the neomycin-resistance gene containing EBV and expresses low level of LMP1. With this cell line it was thus possible to study the regulation of LMP1 expression by modification of chromatin acetylation state. STUDY DESIGN: The TWO-EBV cell line was treated with n -butyrate (NB) or trichostatin A (TSA). RESULTS: Shown by immunoblotting, the LMP1 level was elevated in the treated samples. Already 2 h after TSA exposure LMP1 expression was higher and it increased up to 24 h. Immunofluorescence staining showed that nearly all cells were LMP1 positive. Neither EBNA2 nor BZLF1 were induced. Tested first 2 h after the treatment, acetylated histone H3 and H4 were already detectable, and their level increased up to 8 h. Chromatin immunoprecipitation (ChIP) verified that the LMP1-promoter (LMP1p) (ED-L1) was acetylated after TSA treatment. CONCLUSION: EBV carrying epithelial cells do not express EBNA-2. We showed that LMP1 expression was upregulated by histone deacetylase inhibitors in an in vitro infected, EBV carrier NPC cell line.     

EB病毒潜伏膜蛋白2A基因重组逆转录病毒载体和稳定表达细胞株的构建及鉴定

目的:克隆EB病毒(EBV)潜伏膜蛋白2A(LMP2A)全长基因, 并构建含有该目的基因的重组逆转录病毒载体pGEZ-LMP2A, 转染真核细胞后获得稳定表达LMP2A的细胞株.方法:采用RT-PCR技术从B95.8细胞中获得EB病毒LMP2A全长基因, 克隆到测序载体pMD18-T中, 经测序后再亚克隆到逆转录病毒载体pGEZ-Term中, 与两辅助病毒载体PHIT456、 PHIT60通过Lipofectamine2000共转染包装细胞293T, 测定产生的重组逆转录病毒滴度并感染小鼠成纤维细胞L929, 经Zeocine筛选后得到稳定的细胞克隆, 用RT-PCR和Western blot法检测细胞中LMP2A mRNA和蛋白表达情况.结果:重组逆转录病毒载体pGEZ-LMP2A经测序鉴定正确;转染后上清中病毒的滴度为5×108 cfu/L, 感染L929细胞后用Zeocine筛选出稳定的细胞克隆;RT-PCR和Western blot检测结果表明筛选出的细胞克隆能稳定表达EBV-LMP2A.结论:表达EBV-LMP2A细胞株的构建为蛋白制备与纯化奠定了物质基础, 也为后续的EBV相关性疾病疫苗的研究提供了新思路.     

鼻咽癌中EB病毒编码的潜伏膜蛋白1对β-catenin转录活性及表达的影响

目的 观察鼻咽癌细胞潜伏膜蛋白1(LMP1)表达对β-catenin的转录活性及蛋白表达的影响,探讨LMP1与Wnt/β-catenin信号通路在鼻咽癌发生发展中的作用.方法应用免疫组织化学EnVision法检测75例鼻咽癌组织中LMP1和β-catenin的表达.构建pHA2-LMP1重组质粒,应用细胞免疫荧光、双荧光素酶报告分析、Western blot方法研究LMP1在鼻咽癌细胞株CNE1和CNE2中对β-catenin转录活性及表达的影响.结果 (1)鼻咽癌组织中β-catenin的异常表达率为50.7%(38/75),LMP1的阳性表达率为50.7%(38/75).鼻咽癌组织中β-catenin异常表达与LMP1表达存在正相关(相关系数为x2=7.048,P=0.008).(2)LMP1表达明显提高鼻咽癌细胞株CNE1和CNE2中β-catenin的核表达,且β-catenin的核表达在低分化鼻咽癌细胞株CNE2明显高于高分化鼻咽癌细胞株CNE1.(3)LMP1可明显上调CNE1和CNE2中β-catenin的转录活性,且呈时间依赖性.另外,β-catenin的转录活性在低分化鼻咽癌细胞株CNE2高于高分化鼻咽癌细胞株CNE1.(4)LMP1对鼻咽癌细胞株β3-catenin的总蛋白表达水平无明显影响.结论 EB病毒编码的LMP1可能通过β-catenin信号通路参与鼻咽癌的发生发展.     

EBV-LMP及二甲基亚砜对人鼻咽癌细胞生长分化的影响

目的：研究ＥＢＶ－ＬＭＰ及二甲基亚砜（ＤＭＳＯ）对人鼻咽癌（ＮＰＣ）细胞体外生长分化的影响。方法：以人高分化鼻咽癌细胞株（ＣＮＥ１）为对象，采用电穿孔基因转染技术，将重组ＥＢＶ－ＬＭＰ表达质粒转染ＣＮＥ１细胞，以载体质粒转染及ＣＮＥ１细胞为对照，用细胞体外增殖实验、流式细胞仪和免疫组化等方法，观察细胞生长分化的变化情况。结果：ＥＢＶ－ＬＭＰ在体外可明显促进ＣＮＥ１细胞的增殖，实验组与空白组及阴性对照组比较Ｐ＜０．０１，实验组Ｓ期细胞明显增多，细胞角蛋白表达显著下降（Ｐ＜０．０５），有向低分化鳞癌发展倾向；ＤＭＳＯ诱导后ＣＮＥ１细胞增殖力明显下降（Ｐ＜０．０１），细胞角蛋白表达显著增高（Ｐ＜０．０１）；ＬＭＰ可明显抑制ＤＭＳＯ对ＣＮＥ１细胞的诱导分化作用，转染细胞诱导后增殖力和角蛋白表达无显著改变（Ｐ＞０．０５）。结论：ＤＭＳＯ可在一定程度上诱导ＣＮＥ１细胞分化；ＥＢＶ－ＬＭＰ对ＣＮＥ１细胞生长有明显的促进作用，可抑制细胞分化及降低细胞对终末分化信号的反应，有助于深入研究ＮＰＣ的发生机理及防治     

Antitumor activity of the pachymic acid in nasopharyngeal carcinoma cells

Objective: To investigate the antitumour efficacy of pachymic acid (PA), which is a fungal extract component, on nasopharyngeal carcinoma (NPC) cells CNE-1, CNE-2. Methods: We have chosen NPC cell line CNE-2 for the study, and the cells were treated with PA before the detection. CCK-8 assay was used to detect the proliferative ability, and Annexin V-PI double staining was used for the detection of apoptosis rate; and the nucleus damage was detected by transmission electron microscope, the protein expression of the DNA damage pathway were detected by Western blot. Results: PA can significantly inhibited proliferation of CNE-1, CNE-2 cells. The proportion of apoptotic cells of all cell lines gradually increased in a dose-dependent manner induced by PA, P < 0.05. Meanwhile, the nucleus could be caused morphological changes and the expression of DNA damage-related proteins was upregulated by PA in CNE-2. Conclusions: PA can significantly inhibit cell proliferation and increase the apoptosis rates and may induce the apoptosis of the human NPC cells.     

Alterations of biologic properties and gene expression in nasopharyngeal epithelial cells by the Epstein-Barr virus-encoded latent membrane protein 1

Undifferentiated nasopharyngeal carcinoma (NPC) is closely associated with EBV infection, and the EBV-encoded latent membrane protein 1 (LMP1) is frequently detected in NPC. However, little is known about the pathologic roles of LMP1 in this disease. Recently, we reported the morphologic transformation and increased expression of the LAMC2 and ITGalpha6 genes in LMP1-expressing NPC cell lines. In this study, we further examine the effects of LMP1 in an immortalized nasopharyngeal epithelial cell line called NP69. This cell line was established from primary nonmalignant nasopharyngeal epithelial cells and may represent a model of premalignant nasopharyngeal epithelial cells. LMP1 induced many phenotypic changes in NP69 cells. These include morphologic transformation, increased cell proliferation, anchorage-independent growth, resistance to serum free-induced cell death, and enhanced cell migration and invasion. In addition, expression array analysis identified 28 genes that demonstrated a more than 2-fold difference in expression of NP69 cells expressing LMP1 when compared with a vector control. Two of the up-regulated genes (VEGF and vimentin) identified have been previously reported as LMP1 targets. The majority of the identified genes are associated with cell growth, differentiation, cell shape, and invasion. The present findings support the proposed roles of LMP1 in promoting cell transformation, migration, and invasion in premalignant nasopharyngeal epithelial cells. The present study also indicates the activation of the Ras/MAPK pathway in LMP1-expressing cells, which may be involved in mediating some of the transforming effects of LMP1 observed in nasopharyngeal epithelial cells.