EB病毒miRNAs在鼻咽癌细胞株C666-1和CNE-2Z中的差异性表达分析

目的：探讨ebv-miRNA在低分化NPC细胞株C666-1（EBV＋）和CNE-2Z（EBV表达水平随传代次数增加而逐渐降低）中的差异性表达进而分析其功能.方法：常规培养NPC细胞株C666-1和CNE-2Z,分别提取总RNA并进行质检;RT-PCR检测LMP-1和LMP-2A mRNA的表达以判断EBV的感染情况;miRNA芯片技术检测EBV miRNAs的表达并对其表达谱进行差异性分析;荧光定量RT-PCR进行验证;复习文献了解ebv-miRNA调控的靶基因以了解其功能.结果：从C666-1和CNE-2Z两株细胞抽提的总RNA纯度高,A260/A280〉2.0,A260/A230〉2.1,RNA完整性好.C666-1具有比CNE-2Z更高的LMP-1和LMP-2A mRNA表达.ebv-miRNA表达谱的差异分析表明39个ebv-miRNAs中19个在两株细胞显著差异性表达;荧光定量RT-PCR结果证实ebv-miR-BHRF1-1和ebv-miR-BART14＊在C666-1中表达升高而ebv-miR-BART8＊表达降低,与芯片结果趋势一致;复习文献,EBV miRNAs功能远不清楚,少数已知的靶基因涉及信号转导、转录调控、细胞凋亡、增殖、免疫反应和病毒DNA复制等方面.结论：ebv-miRNA在低分化NPC中具有差异性表达并广泛参与基因调控.不同NPC细胞中EBV miRNA表达差异预示着基于ebv-miRNA的NPC个性化治疗的可能性.     

EB病毒miRNAs在鼻咽癌细胞株C666-1和CNE-2Z中的差异性表达分析

目的:探讨ebv-miRNA在低分化NPC细胞株C666-1(EBV+)和CNE-2Z(EBV表达水平随传代次数增加而逐渐降低)中的差异性表达进而分析其功能.方法:常规培养NPC细胞株C666-1和CNE-2Z,分别提取总RNA并进行质检;RT-PCR检测LMP-1和LMP-2A mRNA的表达以判断EBV的感染情况;miRNA芯片技术检测EBV miRNAs的表达并对其表达谱进行差异性分析;荧光定量RT-PCR进行验证;复习文献了解ebv-miRNA调控的靶基因以了解其功能.结果:从C666-1和CNE-2Z两株细胞抽提的总RNA纯度高,A260/A280>2.0,A260/A230>2.1,RNA完整性好.C666-1具有比CNE-2Z更高的LMP-1和LMP-2A mRNA表达.ebv-miRNA表达谱的差异分析表明39个ebv-miRNAs中19个在两株细胞显著差异性表达;荧光定量RT-PCR结果证实ebv-miR-BHRF1-1和ebv-miR-BART14*在C666-1中表达升高而ebv-miR-BART8*表达降低,与芯片结果趋势一致;复习文献,EBV miRNAs功能远不清楚,少数已知的靶基因涉及信号转导、转录调控、细胞凋亡、增殖、免疫反应和病毒DNA复制等方面.结论:ebv-miRNA在低分化NPC中具有差异性表达并广泛参与基因调控.不同NPC细胞中EBV miRNA表达差异预示着基于ebv-miRNA的NPC个性化治疗的可能性.     

EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响

背景与目的:已证实EB病毒编码的潜伏膜蛋白1(latentmembraneprotein,LMP1)具有转化致瘤作用,在鼻咽癌的发生发展中起重要作用。LMP1的转化致瘤作用可能与细胞凋亡有关,本实验目的是观察EBV-LMP1对鼻咽癌细胞系CNE1细胞凋亡的影响,探索LMP1在鼻咽癌发生发展中的可能机制。方法:用电转染的方法将带有绿色荧光蛋白GFP报道系统的EB病毒LMP1基因真核表达质粒导入CNE1,用荧光显微镜检测报道基因GFP的表达情况;用免疫组化法及Westernblot法检测目的基因LMP1的表达情况;用流式细胞仪、荧光染色及DNA片段分析等方法检测在有地塞米松磷酸钠或无血清饥饿诱导的情况下LMP1对CNE1细胞凋亡的影响。结果:CNE1G细胞(转染空载质粒PAT-GFP的CNE1细胞)及CNE1GL(转染目的基因质粒PAT-GFP-LMP1的CNE1细胞)有绿色荧光蛋白表达,CNE1细胞(未经转染的CNE1细胞)无绿色荧光蛋白表达;CNE1GL细胞LMP1呈阳性表达,而CNE1及CNE1G细胞均为阴性。CNE1、CNE1G及CNE1GL3组细胞分别用地塞米松磷酸钠或无血清1640培养液处理后均有凋亡出现,且两种诱导方法均能使CNE1GL组细胞的凋亡指数(apoptosisindex,AI)较CNE1及CNE1G组明显增高(P<0.05),而CNE1与CNE1G组的凋亡指数无明显差别。3组细胞常规培养下则无凋亡出现。结论:LMP1基因成功导入CNE1细胞     

5-氮杂胞苷增加鼻咽癌细胞株C666-1放射敏感度的实验

 目的 研究5-氮杂胞苷（5-azacytidine, 5-Aza）对人鼻咽癌细胞C666-1放射敏感度的影响及可能机制。方法 用不同浓度5-氮杂胞苷（0、500、1000、3000、5000 nmol/L）处理C666-1细胞,MTT法检测C666-1细胞增殖,流式细胞仪检测C666-1细胞周期,Western blot法检测细胞凋亡相关蛋白cleaved Caspase-3、cleaved PARP及DNA损伤相关蛋白γ-H2AX的表达。结果 5-氮杂胞苷随着药物浓度的增加,对C666-1细胞生长抑制作用增强。用1 000 nmol/L 5-氮杂胞苷处理鼻咽癌细胞进行放射增敏研究。鼻咽癌细胞C666-1接受单纯电离辐射时SF2（照射2 Gy时的细胞存活率）为48%,5-氮杂胞苷联合电离辐射时SF2降为31%,放射增敏比为1.37。5-氮杂胞苷能增加电离辐射诱导凋亡相关蛋白cleaved Caspase-3及cleaved PARP的表达。结论 5-氮杂胞苷增加鼻咽癌C666-1细胞对电离辐射的敏感度,可能的机制为5-氮杂胞苷能增加电离辐射诱导细胞凋亡。     

EBV-LMP1对鼻咽癌细胞系CNE1细胞转移相关因素的影响

背景与目的:已证实EB病毒(Epstein-Barrvirus,EBV)编码的潜伏膜蛋白1(latentmembraneprotein1,LMP1)能够诱导鼻咽癌细胞中基质金属蛋白酶9(matrixmetalloproteinase-9,MMP-9)的表达。本实验的目的是观察EB病毒潜伏膜蛋白1(EBV-LMP1)对鼻咽癌细胞系CNE1细胞转移相关因素的影响,探讨LMP1在鼻咽癌侵袭、转移过程中的作用。方法:用免疫组化法及Westernblot法检测CNE1-GL(转染LMP1基因的CNE1细胞)和CNE1细胞中MMP-9的表达情况;用细胞-基质粘附实验、细胞运动实验和肿瘤细胞重组基底膜侵袭实验检测LMP1对CNE1细胞粘附、运动及侵袭能力的影响。结果:免疫组化法及Westernblot法结果均显示CNE1-GL细胞中MMP-9的表达明显高于CNE1细胞(P<0.05);肿瘤细胞-基质粘附实验结果显示,CNE1-GL的粘附能力(平均吸光度值为1.2508±0.0711),高于CNE1细胞(平均吸光度值为0.9519±0.068),两者相比差异有显著性(P<0.01)。运动实验及重组基底膜侵袭实验结果均显示,穿过游离的聚乙烯吡咯烷酮膜(polyvinylpyrroli-done-free,PVP-F)的CNE1-GL细胞数明显高于CNE1细胞(P<0.01)。结论:LMP1能够诱导CNE1细胞中MMP-9的表达,且增强CNE1细胞与基底膜的粘附能力、运动能力及侵袭能力。     

hTERTC27过表达对鼻咽癌C666-1细胞株增殖和凋亡的影响

目的 研究外源表达hTERTC27对鼻咽癌C666-1细胞增殖和凋亡的影响.方法 采用脂质体将pcDNA3 hTERTC27转染至C666-1细胞株中.采用Western blot法鉴定hTERTC27和Bcl-2在细胞中的表达.采用MTT比色法和细胞克隆形成实验观察其对细胞增殖的影响.采用TUNEL实验观察其对细胞凋亡的影响.结果 脂质体转染24h后,C666-1细胞株外源性hTERTC27出现高表达.过量表达hTERTC27的C666细胞增殖能力明显减缓(P＜0.01),克隆形成率降低(P＜0.01),TUNEL阳性细胞率增加(P＜0.01),Bcl-2表达量降低(P＜0.01).结论 hTERTC27可以抑制C666-1细胞增殖、促进凋亡,可能与下调Bcl-2蛋白的表达有关.     

Expression of the Epstein-Barr virus genome in a nasopharyngeal carcinoma epithelial tumor cell line

An epithelial tumor cell line was recently established from a biopsy specimen of a nasopharyngeal carcinoma (NPC), and designated HONE-I. Uncloned (parental) HONE-I and HONE-I clone (C)-40 cells were found to contain latent Epstein-Barr virus (EBV). Expression of the latent EBV genome in HONE-I C-40 cells has been examined. It was possible to detect a small percentage of cells spontaneously synthesizing EBV early antigen (EA) and virus capsid antigen (VCA) by immunofluorescence (IF). In addition, the EBV nuclear antigens (EBNA-I and EBNA-2), as well as the EBV latent membrane protein (LMP) were detected in the HONE-I cells. Attempts were made to induce the latent EBV genome in these cells with iododeoxyuridine (IUdR). We observed a significant increase in the number of EA/VCA-positive cells, an increase in EBV DNA, the synthesis of virus particles, and the rescue of infectious virus after treatment of HONE-I C-40 cells with IUdR. The HONE-I C-40 cells should facilitate studies of the expression and regulation of the EBV genome in NPC epithelial tumor cells, which have not previously been available.     

EB病毒感染鼻咽癌细胞后的增殖周期变化

背景与目的:EB病毒(Epstein-Barrvirus,EBV)在不同组织类型鼻咽癌细胞内的增殖周期特征决定了基因治疗所采取的策略。本研究探讨EBV感染鼻咽癌细胞后的增殖周期特征。方法:利用EBV阳性的B95-8细胞标准株与鼻咽癌细胞CNE2接触培养,通过补体激活的细胞毒性试验去除共培养体系中的B95-8细胞,并采用逆转录聚合酶链式反应及Southern印迹杂交的方法分析鼻咽癌细胞中病毒的初始感染状态。结果:B95-8细胞与CNE2细胞接触共培养的方法使EBV成功感染CNE2细胞,EBV裂解期标志性基因BZLF1转录时间平均为12d,病毒感染标志性基因EBER转录时间平均为22d。结论:EBV侵入CNE2细胞后可能多数病毒开始处于裂解期增殖,之后EBV从裂解期进入了相对稳定的潜伏期,并随着细胞的传代培养病毒逐渐丢失。     

MALAT-1基因种属同源序列重组质粒的构建及其与人鼻咽癌细胞株C666-1侵袭转移的关系

目的:运用基因重组技术构建人MAL-AT-1基因种属同源序列(Species homologous frag-ments/shf)重组真核荧光表达载体,并探讨其在MALAT-1高表达的人鼻咽癌细胞株C666-1中的表达和对其侵袭转移的影响。方法:应用RT-touchdown PCR的方法,从鼻咽癌患者正常切缘组织中扩增出MALAT-1基因的种属同源序列,将所得的cDNA序列定向克隆至真核表达载体pEGFP-C1质粒中,构建出重组真核荧光表达载体;采用脂质体Lipofectamine 2000介导重组质粒pEGFP-C1-MALAT-1/shf瞬时转染MALAT-1高表达的人鼻咽癌细胞株C666-1;通过实时荧光定量PCR方法检测MALAT-1基因种属同源序列转染组和空载体转染组细胞中MALAT-1/shf的表达水平;细胞基质胶侵袭实验研究转染组细胞侵袭能力的变化。结果:经酶切鉴定及测序分析,证实成功构建人MALAT-1/shf重组真核荧光表达载体。pEGFP-C1-MALAT-1/shf(6 918~8 441 bp)转染组平均侵袭细胞数(51.20±5.933)显著高于MALAT-1/mock组(13.40±3.847),差异具有统计学意义(P=0.000)。这说明转染pEGFP-C1-MALAT-1/shf(6 918~8 441 bp)重组质粒后,细胞的侵袭能力明显增强。结论:成功构建了人MALAT-1基因种属同源序列真核荧光表达载体pEGFP-C1-MALAT-1/shf,并成功在MALAT-1高表达的人鼻咽癌细胞株C666-1中表达,MALAT-1/shf(6 918~8 441 bp)与人鼻咽癌C666-1细胞株的侵袭转移密切相关。     

Detection of Epstein-Barr virus genome in natural-killer-like cell line, YT.

YT cells, originally reported as a natural-killer-like (NK-like) lymphoid cell line, were investigated for Epstein-Barr virus (EBV) genome, gene rearrangement for T-cell receptor (TCR), phenotype and function. The YT cells of the original batch (YT-0) and two subclones (YT2C2 and YTC3) expressed EBV-associated nuclear antigen, and the BamHI-digested DNA showed the 3.4 kb hybridizing band with the BamHIW probe of EBV DNA in Southern blot analysis. When tested with latent-infection membrane protein probe, an identical hybridizing band was shared, indicating that all three sources of YT cells were of monoclonal derivation in terms of the terminal repeat junctional structure of EBV DNA, and that the original YT cells had been infected with EBV before the isolation of the two subclones. The cell-surface antigen analysis revealed the expression of CD7, CD28, CD30, CD45R0, TLiSA and S6F1 antigen besides the originally recorded CD25, CD56 and HLA-DR antigen. Gene rearrangement analysis showed the germ-line genotype, including TCR gamma and delta as well as beta chain. The Northern blot study using the CD3 epsilon and CD3 delta chain gene probes revealed CD3 epsilon, but not CD3 delta RNA. The YT-0 cells exhibited NK and antibody-dependent cellular cytotoxicity activity, but the YT2C2 and YTC3 cells did not. It was not resolved whether the fresh neoplastic NK-like cells of the YT-cell donor carry EBV genome, but YT cells, the first lymphoid cell line found to have EBV genome and non-B lymphoid properties, are valuable for investigating the relationship between EBV and human non-B lymphoid hematopoietic cells.     

Expression of Epstein-Barr virus in renal cell carcinoma

There have been few studies regarding the etiology of renal cell carcinoma. To examine the possible involvement of Epstein-Barr virus (EBV) in this disease, 9 renal cell carcinoma (RCC), 2 nephroblastoma (Wilms' tumor) and 2 RCC cell lines were subjected to mRNA in situ hybridization and indirect immunofluorescence staining. Messenger RNA in situ hybridization using BamHIW, EBNA LP, EBNA 2 and EBER1 probes of EBV revealed signals in all the examined samples, although some samples showed weak signals using the EBNA LP probe. Indirect immunofluorescence staining using anti-EBNA LP, anti-EBNA2, anti-LMP1 and anti-BZLF1 antibodies showed definitive fluorescence. PCR also revealed EBV DNA in all 8 RCC specimens including 7 cases other than hybridization and fluorescence. EBV infected all the RCC and nephroblastoma irrespective of the histological or clinical stage. On the other hand, EBV expression was stronger in papillary and clear cell-type RCC than chromophobe cell-type, as well as being stronger in the higher grades of RCC. These results suggest that the expression of EBV may be involved in the pathogenesis of RCC and nephroblastoma.     

Epstein-Barr virus and nasopharyngeal carcinoma.

Nasopharyngeal carcinoma, an epithelial tumor which is characterized by marked geographic and population differences in incidence, is consistently associated with the Epstein-Barr virus (EBV). Within the tumor, the EBV DNA is homogeneous and clonal with regard to repeat sequences suggesting that the tumor is also clonal. Expression of specific viral mRNAs or gene products are consistently detected within all of the tumor cells. These data suggest that EBV is an essential component in the development of nasopharyngeal carcinoma. Genetic or environmental co-factors may influence the ability of the virus to express these genes in infected epithelial tissue or may contribute to clonal predominance.     

New hepatocellular carcinoma cell line SUHC-1 established from a patient with hepatitis C virus RNA in serum.

A new human hepatocellular carcinoma cell (HCC) line, designated SUHC-1, was derived from a Japanese patient with hepatocellular carcinoma having antibody to hepatitis C virus (HCV) and HCV-RNA in his serum, and established in tissue culture. This cell line exhibited typical epithelial cell morphology in culture as observed by phase-contrast and electron microscopy. The SUHC-1 cells produced albumin and alpha 2-macroglobulin. Chromosomal analysis showed several rearrangements at short and long arms of chromosome 1, 17 and 20 (1p-, 1q-, i(1q), i(17q) and 20q+) with a modal number of 91. HCV-RNA was not detected in the supernatant of SUHC-1 cells by nested polymerase chain reaction assay or in the SUHC-1 cells by the in situ hybridization method. We concluded that complete HCV does not exist in the SUHC-1 cell line. The SUHC-1 cell line is the first line of HCC to have been derived from a patient with persistent HCV infection, and may provide a suitable model for studies of hepatocarcinogenesis related to HCV.     

EB病毒LMP1对鼻咽癌中瘤细胞分化的影响

目的　探讨EB病毒LMP1对早期鼻咽癌中瘤细胞分化的影响。方法　收集 31例早期鼻咽非角化性癌活检组织。采用DAKO公司的PNA探针和PNA原位杂交试剂盒检测EB病毒早期RNAs (EBERs)。应用免疫组化法检测LMP1,α catenin ,β catenin ,γ catenin以及高、低分子量细胞角蛋白。结果　 31例癌组织中均有相当数量的瘤细胞呈EBERs阳性 ,其中19例表达LMP1( 6 1.2 9% )。LMP1阳性组γ catenin的表达百分率 ( 5 3 .2 5± 34.12 % )明显低于LMP1阴性组 ( 80 .42±15 .77% ,P <0 .0 1)。γ catenin的表达与低分子量细胞角蛋白的表达间呈正相关 (r=0 .44 0 ,P <0 .0 5 )。α catenin ,β catenin和γ catenin的表达相互间呈两两正相关。结论　鼻咽癌中瘤细胞的LMP1能下调γ catenin的表达 ,从而抑制瘤细胞的分化。     

姜黄素对鼻咽癌C666-1细胞增殖的抑制作用及其可能机制

观察姜黄素对鼻咽癌细胞株C666-1增殖及凋亡的影响,探讨其可能的作用机制。方法：0、10、25、50及100 μmol/L姜黄素作用24 h或50 μmol/L姜黄素不同时间（0、6、12及24 h）后, CCK-8法检测C666-1细胞的增殖情况,TUNEL法检测细胞凋亡,Western blotting检测不同浓度姜黄素作用24 h后细胞内AMPK、S6K1及S6蛋白磷酸化情况。结果：与0 μmol/L 组比,50、100 μmol/L姜黄素作用24 h后,对C666-1细胞增殖的抑制作用显著增强[（38.33±6.53）%、（2114±5.36）% vs （100±0.00）%, 均P<0.05];50 μmol/L姜黄素作用6 、12 、24 h后,对C666-1细胞增殖的抑制作用显著增强[（49.6±5.67）%、（47.7±6.65）%、（46.86±9.4）% vs （100±0.00）%,均P<0.05]。50、100 μmol/L姜黄素作用后细胞的凋亡率显著增加[（43±12.53）%、（48±8.54）% vs （2.87±1.03)%,均P<0.05],50 μmol/L姜黄素作用12 、24 h后,细胞凋亡率随作用时间延长而增加[（35.33±5.86）%、（47.33±13.01）% vs (4.33±3.21)%,均P<0.05]。50、100 μmol/L姜黄素可促进细胞内AMPK磷酸化,抑制其下游mTOR信号通路中S6K及S6蛋白的磷酸化活化[（3.87±1.38）、（019±0.16）、（0.39±0.24） vs 1; (4.34±1.34)、(0.059±0.043)、(0.11±0.095) vs 1,均P<0.05]。结论：姜黄素可显著抑制鼻咽癌C666-1细胞增殖和诱导细胞凋亡,其机制可能与影响AMPK、S6K1及S6蛋白磷酸化有关。     

鼻咽癌细胞株裸鼠移植瘤中癌细胞的凋亡

观察鼻咽癌细胞株ＣＮＥ－１和ＣＮＥ－２裸鼠移植瘤组织中肿瘤细胞凋亡的形态学特征,并探讨ｐ５３,ｂｃｌ－２和ｂａｘ在瘤细胞凋亡中所起的作用。方法：ＣＮＥ－１和ＣＮＥ－２鼻咽癌细胞株接种于裸鼠并在裸鼠体内连续传３代。     

高迁移率族蛋白1对人鼻咽癌细胞株C666-1体外迁移及侵袭能力的影响

目的 观察高迁移率族蛋白1（HMGB1）对人鼻咽癌细胞株C666-1体外迁移及侵袭能力的影响,并初步探讨其可能的机制。 方法 用siRNA干扰C666-1细胞株HMGB1基因表达,通过划痕试验及Transwell检测siHMGB1对C666-1细胞株迁移及侵袭能力的影响,通过Western blot检测HMGB1干扰后侵袭相关蛋白基质金属蛋白酶2/9（MMP2/9）的表达变化。结果 相比对照组,干扰C666-1细胞株HMGB1基因的表达后,C666-1细胞迁移能力明显下降（P<0.001）, 侵袭能力明显减弱（P<0.001）,细胞侵袭相关蛋白MMP2表达水平明显下降（P<0.001）,而MMP9表达未见明显变化。结论 HMGB1促进人鼻咽癌细胞株C666-1体外迁移及侵袭,且其对C666-1侵袭能力的影响可能通过MMP2介导。     

Tumorigenicity of Epstein-Barr virus (EBV)-transformed lymphoid line cells in autologous squirrel monkeys.

Eight squirrel monkeys (Saimiri sciureus) challenged with EBV or EBV-transformed SLCL were naturally or experimentally infected with Plasmodium knowlesi or Pl. brasilianum. Most of the animals had been splenectomized and unilaterally nephrectomized. Three of these monkeys received one dose of 6 to 12 X 10(8) autologous SLCL. These lines were derived from saimiri lymphoid cells permanently transformed by B-EBV in vitro. All three animals developed multiple undifferentiated malignant lymphomas and died 8 to 10 days post inoculation. Necropsy tumor specimens were EBNA-positive and contained 7 to 21 EBV genome equivalents per cell. EBNA- and EA-positive SLCL were established in vitro from four tumor explants of two monkeys. These results demonstrate that in vitro EBV-transformed SLCL are able to cause tumor formation in autologous squirrel monkeys. Five monkeys received high transforming doses of B-EBV or S-EBV, derived from one of the tumorigenic SLCL. None of the animals developed any sign of tumor formation during an observation period of up to 130 days. Three of these monkeys showed no detectable EBV-related seroconversion while the sera of two monkeys had become anti-EBNA-positive when tested at days 28 and 130 respectively post inoculation. Two additional monkeys received neither EBV nor SLCL. They showed no clinical evidence of tumor development or spontaneous seroconversion over a period of more than 1 year.