EBV阳性胃癌细胞系与阴性胃癌细胞系全基因组表达谱差异比较（英文）

目的:探讨EB病毒阳性胃癌细胞系与EB病毒阴性胃癌细胞系全基因组表达谱的差异。方法:利用Agilent人类全基因组表达谱芯片技术在3种EB病毒阳性胃癌细胞系和3种EB病毒阴性胃癌细胞系中筛选EB病毒相关胃癌肿瘤相关基因。选取6条差异表达基因,应用实时荧光定量PCR验证芯片结果的可靠性。结果:聚类热图及矩阵图分析显示,EB病毒阳性胃癌细胞系与阴性胃癌细胞系之间表达谱存在明显差异。差异表达基因涉及多种生物学过程。选取的6条差异基因进行验证,其表达趋势与芯片结果一致。结论:感染EB病毒可能会改变肿瘤相关基因的表达,进而在EB病毒相关胃癌的发生、发展及预后中发挥作用。筛选出的肿瘤相关基因涉及多种生物学过程,提示多基因及相关基因的变异参与了EB病毒相关胃癌的形成。     

利用表达谱基因芯片筛选溃疡性结肠炎差异表达基因

目的:利用人类全基因组表达谱芯片技术,分析溃疡性结肠炎患者和健康者基因表达谱差异,筛选出溃疡性结肠炎相关基因。方法:采用Trizol法提取8例溃疡性结肠炎患者和8例健康对照者结肠粘膜组织总RNA并纯化,逆转录合成c DNA,利用荧光染料Cy3标记aa UTP,转录合成标记的c RNA,并与Agilent人类全基因组表达谱芯片杂交,扫描荧光信号图像,对芯片原始数据进行归一化处理,利用倍数差异和t检验计算筛选出相关差异表达基因,采用DAVID在线分析系统进行基因的功能注释和关联分析,明确差异基因的生物学功能,并对部分差异表达基因进行实时荧光定量PCR验证。结果:筛查出溃疡性结肠炎结肠粘膜组织差异表达基因4132个,其中上调基因2004个,下调基因2128个。选取6条差异表达基因进行PCR验证,结果有3条基因表达上调,3条基因表达下调,表达趋势与芯片结果一致。结论:溃疡性结肠炎患者与健康对照者基因表达存在明显差异,分析这些差异表达基因有助于我们探索溃疡性结肠炎的发病机制,为疾病的治疗提供理论依据。     

人胃癌细胞系中FHIT基因mRNA的表达及pcDNA3．1-FHIT的构建与转染

目的:检测人胃癌细胞系中FHIT基因mRNA的表达状况及构建pcDNA3.1-FI-IIT真核表达载体.方法:RT-PCR法检测三种不同类型人胃癌细胞系中FHIT基因mRNA的表达,构建真核表达质粒pcDNA3.1-FHIT,通过酶切法、PCR扩增法和DNA序列分析鉴定重组质粒后,用脂质体转染至FHIT基因mR.NA阴性表达人胃癌细胞系MKN-45,经G418筛选后RT-PCR鉴定.结果:FHIT基因在人胃癌细胞系BGC-823中呈阳性表达,在MGC-803、MKN-45细胞系中呈阴性表达.FHIT基因cDNA正确克隆到真核细胞表达栽体pcDNA3.1中,并成功转染FHIT基因mRNA阴性表达人胃癌细胞系MKN-45.结论:FHIT基因在不同类型人胃癌细胞系中表达各异.成功构建pcDNA3.1-FHIT,并转染到FHIT基因mRNA阴性表达人胃癌细胞系MKN-45,使其唧T基因阳性表达.     

猪链球菌2型全基因组DNA芯片的研制及基因表达谱技术平台的建立

目的：研制猪链球菌2型（SS2）全基因组DNA芯片,建立SS2基因表达谱技术平台。方法：利用SS2全基因组序列,挑选出2194条基因,经PCR扩增出2156条基因并将产物纯化,点样制备芯片;将芯片用于表达谱研究,采用实时定量PCR验证表达谱结果,对芯片进行可靠性分析。结果：芯片杂交数据与实时定量PCR验证显示了较高的相关性,二者相关系数r=0.87。结论：研制了一批SS2全基因组DNA芯片,并建立了基于DNA芯片的表达谱技术平台。     

siRNA介导的PRR11表达抑制导致肺癌细胞系基因表达谱变化的分析

Proline rich 11(PRR11)是本课题组鉴定的一个新的肿瘤相关基因。为研究PRR11介导肺癌发生发展相关的分子机制,本研究分析了PRR11表达被抑制后人肺癌细胞系H1299的全基因组基因表达谱的变化。首先,采用siRNA抑制H1299细胞中PRR11的表达,提取总RNA,采用基因芯片分析全基因组基因表达谱的变化。然后,对呈现差异表达的基因进行GO和Pathway富集分析,并对部分重要的候选基因进行定量RT-PCR验证。基因芯片结果表明,采用siRNA有效抑制H1299细胞中PRR11表达后,共有550个基因的mRNA水平出现明显变化,其中139个基因表达上调,411个基因表达下调。生物信息学分析结果表明,上述差异表达的基因显著富集于细胞周期和MAPK通路。定量RT-PCR验证分析结果表明,PRR11表达抑制后确实可导致多个与细胞周期和肿瘤发生发展密切相关的基因(包括DHRS2、EPB41L3、CCNA1、MAP4K4、RRM1、NFIB)呈现显著的表达变化。这些结果提示,PRR11可能通过上述通路和/或基因的表达变化参与肺癌的发生发展过程。     

稳定表达HLA-A33蛋白细胞系的建立

旨在构建能稳定表达HLA-A33蛋白的细胞系,并观察其在细胞中的表达水平.首先克隆取得HLA-A33基因,并将其插入慢病毒载体,经酶切和测序鉴定,确定载体构建正确.通过慢病毒系统感染正常RD细胞,将HLA-A33基因整合进RD细胞的基因组.提取构建细胞系的基因组做PCR鉴定,并通过免疫荧光和蛋白免疫印迹法检测,结果显示,HLA-A33基因成功整合入RD细胞基因组中,且在重组细胞系中成功表达.该细胞系可为A33等位基因与HBV感染后的慢性化以及EV71感染的相关研究提供试验参考.     

用基因芯片筛选前列腺癌LNCaP和C4-2细胞系中差异表达的基因

目的：筛选与前列腺癌进展相关的功能基因。方法：利用Affymetrix公司的人类金基因组U133A芯片,筛选在前列腺癌细胞系LNCaP和C4-2中差异表达的基因,并用RT-PCR方法验证部分差异基因的表达。结果：芯片筛选结果表明,与LNCaP细胞系相比,C4-2细胞系中217个基因转录本表达上调,101个基因转录本表达下调;对其中45个基因进行了RT-PCR验证,证明35个基因转录本的表达结果与芯片筛选一致。结论：筛选出了在LNCaP和C4-2细胞系中显著差异表达的基因35个,为进一步研究前列腺癌进展的机制奠定了基础。     

胃癌多药耐药相关microRNA的筛选和鉴定

目的:研究胃癌多药耐药相关microRNA并对其进行鉴定、靶基因预测和预测靶基因的生物信息学分析。方法:运用microRNA芯片对胃癌多药耐药细胞SGC7901/ADR和其亲本细胞SGC7901进行microRNA表达谱分析;采用实时定量PCR的方法对差异表达的miRNA进行验证;再运用生物信息学方法对差异表达的miRNA进行靶基因预测;再对预测的靶基因进行GO和KEGG通路分析。结果:与SGC7901相比SGC7901/ADR表达上调超过2倍的miRNA有6个,表达下调超过2倍的有11个。实时定量PCR对共同差异表达的microRNA进行验证显示与芯片结果的一致性。对这17个差异表达的miRNA进行靶基因预测,再对预测得到的靶基因进行GO和KEGG通路分析显示预测的靶基因参与了肿瘤相关通路、MAPK通路、Focal Adhesion通路等。结论:我们初步筛选得到了胃癌多药耐药相关miRNA并对其进行了生物信息学分析,为进一步地探索miRNA在胃癌多药耐药中的作用及其分子机制奠定了基础。     

胃癌组织中Bmi-1基因表达的研究

目的：多梳基因家族的Bmi．1被认为是一种癌基因,在多种肿瘤组织中均有表达。本研究主要是检测Bmi-1基因在胃癌组织中的表达及探讨其临床意义。方法：应用RT—PCR和Westernblotting方法检测45例胃癌中Bmi-1基因的表达情况,并结合患者的临床病理资料分析与其相关性。结果：Bmi-1基因阳性表达率在胃癌中为88．9％（40／45）,癌旁组织为17．7％（8／45）,差异有显著性（P〈0．05）;Bmi-1基因阳性表达率与患者年龄、性别、肿瘤大小及有无淋巴结转移无关,与肿瘤分化程度及TNM分期有关（P〈0．05）。结论：Bmi-1基因在胃癌中高表达,与胃癌的疾病进展密切相关,检测Bmi-1的表达可作为胃癌生物学行为的一项评估指标。     

卵巢癌肝转移灶高表达基因SFT2D1的生物信息学分析

目的：研究肿瘤原发灶和转移灶的基因表达差异,并采用生物信息学方法对一条卵巢癌肝转移灶高表达基因SFT2D1进行初步分析。方法：分别将卵巢癌原发灶和肝转移灶组织标本mRNA用Cy3-dUTP和cy5-dUTP标记后与表达谱芯片杂交,通过信号扫描、处理后获得两者的表达差异基因。并用生物信息学方法对一条无功能研究的新基因SFT2D1进行初步分析,阐明了它的基因姑构、染色体定位、编码蛋白质的理化性质、亚细胞定位、蛋白质功能域等信息。并对多物种中的相似性蛋白进行了系统进化分析。结果：表达谱芯片发现了共272条差异表达基因。对新基因SFT2D1的上述性质进行了有效的预测,基本明确了该基因编码蛋白为一内质网跨膜蛋白,可能参与肿瘤转移相关蛋白的合成与加工。结论：表达谱芯片技术是一种研究肿瘤转移基因表达差异的有效的高通量研究方法。通过生物信息学分析,表明新基因SFT2D1是一个有肿瘤转移研究价值的新靶点。     

The oncogenic role of Epstein–Barr virus‐encoded microRNAs in Epstein–Barr virus‐associated gastric carcinoma

Epstein–Barr virus (EBV) infection is detected in various epithelial malignancies, such as nasopharyngeal carcinoma (NPC) and gastric cancer (GC). EBV comprises some unique molecular features and encodes viral genes and microRNAs (miRNAs) by its own DNA sequence. EBV genes are required to maintain latency and contribute to oncogenic property. miRNAs encoded by EBV have been shown to contribute to initiation and progression of EBV‐related malignancies. By a number of genomic profiling studies, some EBV miRNAs were confirmed to be highly expressed in EBV‐associated gastric cancer (EBVaGC) samples and cell lines. The majority host targets of the EBV miRNAs are important for promoting cell growth and inhibiting apoptosis, facilitating cell survival and immune evasion. However, the integrated molecular mechanisms related to EBV miRNAs remain to be investigated. In this review, we summarized the crucial role of EBV miRNAs in epithelial malignancies, especially in EBVaGC. Collectively, EBV miRNAs play a significant role in the viral and host gene regulation network. Understanding the comprehensive potential targets and relevant functions of EBV miRNAs in gastric carcinogenesis might provide better clinical translation.     

Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.     

Expression of viral microRNAs in Epstein-Barr virus-associated gastric carcinoma

Epstein-Barr virus (EBV) is associated with about 6 to 16% of gastric carcinoma cases worldwide. Expression of the EBV microRNAs (miRNAs) was observed in B cells and nasopharyngeal carcinoma cells infected with EBV. However, it is not clear if the EBV miRNAs are expressed in EBV-associated gastric carcinomas (EBVaGCs). We found that BART miRNAs but not BHRF1 miRNAs were expressed in EBV-infected gastric carcinoma cell lines and the tumor tissues from patients as well as the animal model. The expression of viral miRNAs in EBVaGCs suggests that these EBV miRNAs may play important roles in the tumorigenesis of EBVaGCs.     

EB病毒相关胃癌中病毒分型的研究

收集236例胃癌组织标本及135份健康人群咽漱液(throat washings,TWs)标本,采用原位杂交、PCR-Southern blot筛选出17例EB病毒相关胃癌(EBVaGC)(7.2%)和33例EBV阳性的TWs标本(24.4%);应用PCR-RFLP、巢式PCR及DNA测序等方法,检测EBV阳性标本病毒1/2分型、F/f分型、I/i分型及LMP1XhoI(+)/(-)等四种基因变异。EBVaGC及健康对照均为F型变异,未检测到f型变异。EBVaGC中1/2型、I/i型及LMP1XhoI(+)/(-)型的例数及比例分别为:17(100%)/0(0)、1(5.9%)/16(94.1%)及0(0)/15(88.2%);而TWs中上述分型的相应数据为25(75.8%)/8(24.2%)、11(33.3%)/19(57.6%)及12(36.4%)/18(54.5%),各位点两种基因型在EBVaGC和健康人中的分布不同(1/2:P=0.047;I/i:P=0.048;XhoI(+)/(-):P=0.012)。综合分析表明在3种基因多态性均能确定的标本中,EBVaGC均为1/i/XhoI(-)亚型(15/1...     

Sequence variations of latent membrane protein 2A in Epstein-Barr virus-associated gastric carcinomas from Guangzhou, southern China

Latent membrane protein 2A (LMP2A), expressed in most Epstein-Barr virus (EBV)-associated malignancies, has been demonstrated to be responsible for the maintenance of latent infection and epithelial cell transformation. Besides, it could also act as the target for a CTL-based therapy for EBV-associated malignancies. In the present study, sequence variations of LMP2A in EBV-associated gastric carcinoma (EBVaGC) and healthy EBV carriers from Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were investigated. Widespread sequence variations in the LMP2A gene were found, with no sequence identical to the B95.8 prototype. No consistent mutation was detected in all isolates. The immunoreceptor tyrosine-based activation motif (ITAM) and PY motifs in the amino terminus of LMP2A were strictly conserved, suggesting their important roles in virus infection; while 8 of the 17 identified CTL epitopes in the transmembrane region of LMP2A were affected by at least one point mutation, which may implicate that the effect of LMP2A polymorphisms should be considered when LMP2A-targeted immunotherapy is conducted. The polymorphisms of LMP2A in EBVaGC in gastric remnant carcinoma (GRC) were for the first time investigated in the world. The LMP2A sequence variations in EBVaGC in GRC were somewhat different from those in EBVaGC in conventional gastric carcinoma. The sequence variations of LMP2A in EBVaGC were similar to those in throat washing of healthy EBV carriers, indicating that these variations are due to geographic-associated polymorphisms rather than EBVaGC-associated mutations. This, to our best knowledge, is the first detailed investigation of LMP2A polymorphisms in EBVaGC in Guangzhou, southern China, where NPC is endemic.     

Variations of Epstein-Barr Virus Nuclear Antigen 1 in Epstein-Barr Virus-Associated Gastric Carcinomas from Guangzhou,Southern China

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein consistently expressed in all EBV-associated malignancies, and play a critical role in the onset, progression, and/or maintenance of these tumors. Based on the signature changes at amino acid residue 487, EBNA1 is classified into five distinct subtypes: P-ala, P-thr, V-leu, V-val and V-pro. In the present study, the sequence variations of EBNA1 in EBV-associated gastric carcinoma (EBVaGC) and throat washing (TW) samples of healthy EBV carriers in Guangzhou, southern China, where nasopharyngeal carcinoma (NPC) is endemic, were analyzed by PCR and DNA sequencing. V-val subtype was the most predominant (53.6%, 15/28) in EBVaGC, followed by P-ala (42.9%, 12/28) and V-leu (32.1%, 9/28) subtypes. In TWs of healthy EBV carriers, V-val subtype was also predominant (85.7%, 18/21). The sequence variations of EBNA1 in EBVaGC were similar to those in TW of healthy EBV carriers (p>0.05), suggesting that the EBV strains in EBVaGC might originate from the viral strains prevalent within the background population. The predominance of V-val subtype in EBVaGC in Guangzhou was similar to that in EBVaGC in northern China and Japan, but was different from that in EBVaGC in America, suggesting that the variations of EBNA1 in EBVaGC represent geographic-associated polymorphisms rather than tumor-specific mutations. In addition, the EBNA1 variations in EBVaGC in gastric remnant carcinoma were also determined. V-leu subtype was detected in all 4 (100%) cases, although 2 cases occurred as mixed infection with P-ala subtype. This is different from the predominant V-val subtype in EBVaGC in conventional gastric carcinoma, suggesting that V-leu might be a subtype that adapts particularly well to the microenvironment within the gastric stump and enters the remnant gastric mucosa epithelia easily. This, to our best knowledge, is the first investigation of EBNA1 polymorphisms in EBVaGC from endemic area of NPC.     

EBV编码miRNAs在病毒感染和肿瘤发生中的功能和意义

EB病毒(Epstein-Barr virus,EBV)是一种172 kb大小的线性双链DNA病毒,与鼻咽癌、淋巴瘤、胃癌等恶性肿瘤的发生密切相关. EBV编码的微小RNAs (miRNAs)可以调节病毒和宿主细胞基因的表达,并且在癌症发生发展中起着多种作用.本文综述了EBV编码的miRNAs (EBV-encoded miRNA,EBV miRNAs)在病毒感染和肿瘤发生、侵袭转移、抗凋亡、信号通路等方面的生物学功能,以及对于EBV相关肿瘤诊断标志物的潜在意义. EB病毒编码的miRNAs也可能成为进一步研究EBV相关肿瘤治疗的一个候选靶点.     

Epstein-Barr virus infection of human gastric carcinoma cells: implication of the existence of a new virus receptor different from CD21.

Recombinant Epstein-Barr virus (EBV) with a selectable marker successfully infected the human gastric carcinoma cell lines AGS, MKN28, and MKN74. Following incubation in selective media, drug-resistant cell clones were isolated and proved to be infected with EBV. All gastric carcinoma cell clones were positive for EBNA 1 but negative for EBNA 2. LMP 1 expression was negative in most clones, but there were a few exceptions. Gastric carcinoma cells were negative for the EBV receptor CD21, and infection was not inhibited by pretreatment of cells with the anti-CD21 monoclonal antibody OKB7. The results indicate that gastric carcinoma cells are susceptible to EBV infection and that infection is mediated via a new receptor different from CD21.     

Restricted expression of EBV latent genes and T-lymphocyte-detected membrane antigen in Burkitt''s lymphoma cells.

Certain newly established Epstein-Barr virus-containing Burkitt's lymphoma cell lines do not express the cytotoxic T-lymphocyte-detected membrane antigen (LYDMA) through which EBV infection is normally controlled by the host. When the EB virus recovered from these BL lines was used to transform peripheral blood lymphocytes from seronegative donors, the lymphoblastoid cell lines (LCLs) that arose were all LYDMA positive. This indicates that the LYDMA-negative nature of the BLs is not the result of a mutation in the resident viral genome but is rather a specific adaptation in those cells, perhaps permitting evasion of the host immune surveillance in tumour development. A comparison of the EBV gene expression in six LYDMA-negative and two LYDMA-positive BL lines and in their corresponding LCLs revealed that several of the BL lines did not express all of the viral gene products classically associated with latent transformation by EBV. Four out of eight cell lines showed restricted expression of the latent membrane protein (LMP) and/or the EB nuclear antigen, EBNA 2. A new level of EBV gene regulation therefore appears to be operating in some of the BL cell lines. The patterns of expression of EBV genes in the cell lines did not show any correlation with the known susceptibility of the lines to T cell killing.