胃癌患者原癌基因和抑癌基因的表达谱研究

目的：用基因表达谱苡片对人正常胃和胃癌组织原癌和抑癌基因表达的差异进行研究比较。方法：利用胃和胃癌组织的mRNA通过逆转录方法，将Cy3和Cy52种荧光分别标记到2组cDNA上制成探针，然后和表达谱芯片进行杂交后扫描，通过计算机分析判定2种基因是否在上述组织中存在差异表达。结果：在195条原癌及抑癌基因中，存在差异表达的共13条，表达上调的6条（0.071%)。表达下调的7条（0.083%)。结论：认为应用这种方法识别出的2种基因对胃癌的诊断和治疗具有重要的潜在价值。     

人胃腺癌BGC823细胞Midkine高表达前后基因表达谱芯片分析

目的：用基因芯片技术分析、筛选Midkine高表达前后胃腺癌相关基因谱的差异表达。方法：建立Midkine高表达BGC823胃腺癌细胞系,提取细胞总RNA,逆转录Cy3、Cy5标记cDNA探针,应用晶芯唧表达谱芯片,分析差异表达的基因。结果：发现Midkine高表达前后BGC823胃腺癌细胞有显著性表达差异的基因共有550个,其中上调基因407个,下调基因143个。对550条差异表达基因进行初步功能分类分析,结果表明这些基因与Midkine促进胃腺癌发病机制存在相关性。结论：Midkine高表达前后BGC823胃腺癌细胞部分功能基因存在显著的表达差异,多数差异表达的基因参与细胞黏附、细胞骨架形成、细胞周期以及细胞代谢等过程。     

胃癌患者原癌基因和抑癌基因的表达谱研究

目的 :用基因表达谱芯片对人正常胃和胃癌组织原癌和抑癌基因表达的差异进行研究比较。方法 :利用胃和胃癌组织的mRNA通过逆转录方法 ,将Cy3和Cy5 2种荧光分别标记到 2组cDNA上制成探针 ,然后和表达谱芯片进行杂交后扫描 ,通过计算机分析判定 2种基因是否在上述组织中存在差异表达。结果 :在 195条原癌及抑癌基因中 ,存在差异表达的共 13条 ,表达上调的 6条 (0 0 71% ) ,表达下调的 7条 (0 0 83% )。结论 :认为应用这种方法识别出的 2种基因对胃癌的诊断和治疗具有重要的潜在价值     

应用基因表达谱芯片研究人胃癌细胞周期和细胞凋亡相关基因

目的：筛选胃癌发生过程中与正常组织差异表达的细胞周期和细胞凋亡相关基因并作初步功能分析.方法：应用Trizol一步法抽提胃癌及正常组织总RNA,分离纯化mRNA并逆转录合成荧光分子（Cy3/Cy5）标记cDNA探针,与含有8 464种cDNA基因的表达谱芯片杂交,分析胃癌与正常组织间差异表达的基因,对所获得的基因进行分子生物信息学分析.结果：胃癌与正常组织间差异表达的细胞周期和细胞凋亡基因有17条,占芯片基因总数的0.2%.其中与细胞周期相关的基因11条,与细胞凋亡相关的基因6条.在胃癌组织中表达量增加的10条,表达量下降的7条.生物信息学分析显示,该17条差异表达的细胞周期和细胞凋亡相关基因,与胃癌组织细胞的生长和凋亡有关.结论：胃癌的发生、发展过程中存在多基因表达调控的改变,细胞周期和细胞凋亡基因与胃癌组织细胞的生长和凋亡有相关性,对于该相关基因群的进一步研究有助于阐释胃癌的发病机制.     

雷公藤内酯醇对胰腺癌细胞株SW1990基因表达谱的影响

目的:应用基因表达谱芯片研究胰腺癌细胞株(SW1990)在雷公藤内酯醇作用后其基因表达的差异性。方法:用Cy5和Cy3两种荧光染料通过逆转录反应将加药组和对照组的SW1990细胞的mRNA分别标记成两种探针,并与载有一组靶基因的表达谱芯片杂交,通过扫描荧光强度,计算机软件分析,寻找经雷公藤内酯醇作用后表达有差异的基因。对芯片结果中两个表达有改变的基因行荧光定量PCR验证。结果:胰腺癌细胞SW1990受雷公藤内酯醇作用4h后,共筛选出11条差异表达基因,且全部下调。结论:雷公藤内酯醇可能部分通过下调C-MYC、ETS2、TGIF、RTP801等基因来发挥其有效的抗肿瘤特性。     

人胃腺癌BGC823细胞Midkine高表达前后基因表达谱芯片分析

目的:用基因芯片技术分析、筛选Midkine高表达前后胃腺癌相关基因谱的差异表达.方法:建立Midkine高表达BGC823胃腺癌细胞系,提取细胞总RNA,逆转录Cy3、Cy5 标记cDNA 探针,应用晶芯表达谱芯片,分析差异表达的基因.结果:发现Midkine高表达前后BGC823胃腺癌细胞有显著性表达差异的基因共有550个,其中上调基因407个,下调基因143个.对550条差异表达基因进行初步功能分类分析,结果表明这些基因与Midkine促进胃腺癌发病机制存在相关性.结论: Midkine高表达前后BGC823胃腺癌细胞部分功能基因存在显著的表达差异,多数差异表达的基因参与细胞黏附、细胞骨架形成、细胞周期以及细胞代谢等过程.     

人卵巢浆液性乳头状囊腺癌相关基因表达谱

[目的]探讨人卵巢上皮性癌与正常卵巢组织基因表达谱差异。[方法]采用基因芯片技术，按一步法分别抽提10例卵巢浆液性乳头状囊腺癌患者的癌组织和正常卵巢组织的总RNA并纯化mRNA；逆转录合成以Cy5荧光标记卵巢癌细胞和Cy3荧光标记正常卵巢上皮细胞的cDNA作为探针，在含有8464点人类体细胞基因模板的表达谱cDNA芯片上进行杂交。用荧光扫描仪扫描芯片荧光信号图像，用计算机软件对扫描图像进行数字化处理分析。[结果]卵巢浆液性乳头状囊腺癌与正常卵巢上皮比较，差异5倍以上共有185个基因，其中表达上调（其荧光强度增强〉5）有86个，表达下调（其荧光强度减弱〈0.2）有99个。[结论]卵巢上皮性癌与正常卵巢上皮比较差异表达5倍以上的185个基因，可能与卵巢上皮癌的发生和发展有关。     

外源性FHIT基因对多柔比星诱导胃癌MGC-803细胞凋亡的影响

目的：将脆性组氨酸三联体（fragile histidine triad,FHIT）基因导入该基因表达缺失的人胃癌细胞株MGC-803,探讨FHIT基因表达对多柔比星诱导胃癌细胞凋亡的影响。方法：将载有人外源性FHIT基因的真核表达质粒pRcCMV-FHIT用脂质体介导转入FHIT表达缺失的胃癌细胞MGC-803,筛选阳性克隆,同时以空载体pRcCMV转染的胃癌细胞及胃癌细胞株作为对照;以多柔比星作用于3组细胞,用MTT法测定细胞增殖的抑制率,倒置显微镜下观察细胞形态变化,用丫啶橙荧光染色与流式细胞术检测多柔比星处理前后各组胃癌细胞的凋亡率,流式细胞术检测细胞周期的变化。结果：通过流式细胞术检测,经多柔比星处理后,转染FHIT基因的MGC-803细胞凋亡水平（40.66%）与空质粒转染细胞（13.94%）及胃癌细胞（15.81%）相比明显增高,存在显著性差异（P〈0.01）,FHIT基因与多柔比星有轻度的协同促凋亡作用（P〈0.05）;同时FHIT基因转染后的胃癌细胞生长周期出现了明显的G0/G1期阻滞（74.43%vs56.30%、52.30%）;丫啶橙染色亦见转染FHIT基因的胃癌细胞凋亡数明显增多,且细胞的增殖抑制率存在浓度和时间依赖性。结论：外源性FHIT基因表达与多柔比星协同促进胃癌MGC-803细胞凋亡,FHIT基因可提高胃癌细胞对多柔比星的敏感性。     

食管癌和癌旁上皮基因表达谱差异

目的：用基因芯片技术研究食管癌和癌旁组织基因表达谱差异,筛选与早期癌变相关的基因,方法：分别抽提食管癌组织,癌旁组织和正常食管上皮的总RNA并纯化mRNA,将各mRNA逆转录合成以Cy5和Cy3标记的cDNA-一链做探针,分别混合后在2张含有4096条双点人类全长基因的芯片上进行杂交,用扫描仪扫描芯片荧光信号图像,用软件对扫描图像进行数字上处理和分析,结果：食管癌与正常食管上皮比较差异2倍以上共有135个基因,癌旁上皮与正常食管上皮比较差异2倍以上共有31个基因,其中13个基因与食管癌中出现相同,结论：通过三者的基因谱平行比较提示食管癌与正常食管上此比较差异2倍以上的135个基因可能与食管癌的发生和发展有关,癌旁上皮与正常食管上皮比较差异2倍以上的31个基因可能与早期食管癌变的启动和演化有关。     

HDAC抑制剂TSA增强卵巢癌细胞株A2780对腺病毒基因转染效率的体外研究

背景与目的:腺病毒感染依赖于靶细胞表面柯萨奇-腺病毒受体(Coxsackie and adenovirus receptor,CAR)的表达,肿瘤细胞CAR表达的缺失和下调是造成腺病毒为载体基因治疗效果不佳的根本原因。本研究通过观察组氨酸去乙酰化酶(histone deacetylase,HDAC)抑制剂TrichostatinA(TSA)对卵巢癌细胞株A2780CAR表达水平的影响,探讨HDAC抑制剂在腺病毒载体基因治疗中应用的可能性。方法:分别在TSA作用A2780细胞前后检测CARmRNA和蛋白水平的表达,同时采用流式细胞术测定病毒转染效率,MTT实验评价腺病毒携带的胸苷激酶(ADV/TK)的体外抗瘤效应。结果:TSA处理后,A2780细胞内CARmRNA和蛋白表达明显增高。通过流式细胞仪分析GFP/ADV转染后GFP的表达发现,未处理组细胞转染率为(1.24±0.14)%;5nmol/L和100nmol/LTSA处理48h后,细胞的转染效率分别为(7.58±0.32)%和(7.94±0.28)%。MTT结果表明,ADV/TK介导的体外杀伤作用在5nmol/L和100nmol/LTSA处理组较未处理组增强了4～10倍。结论:TSA可以增强针对卵巢癌细胞的腺病毒基因转染效率,为增强卵巢癌基因治疗效果提供可能的方法。     

组蛋白去乙酰化酶抑制剂提高腺病毒对K562细胞转染及杀伤效率的研究

 目的　研究通过观察组蛋白去乙酰化酶（histone deacetylase,HDAC）抑制剂曲古抑菌素A（trichostatin A,TSA）对人类白血病细胞系K562 CAR表达水平的影响,并探讨HDAC抑制剂在以腺病毒为载体的基因治疗中的应用价值。方法　在mRNA和蛋白水平检测TSA处理K562细胞前后CAR的表达情况,采用流式细胞术测定病毒的转染效率,四甲基偶氮唑蓝（MTT）法检测腺病毒及其携带的基因的体外抗瘤效应。结果　TSA处理后,K562细胞CAR 的mRNA 和蛋白表达明显增高;流式细胞仪分析ADV-GFP转染K562 后GFP的表达发现：未处理组的转染率为（8.74±0.34）％,1、10 和100 nmol／L TSA处理细胞48 h后的转染率分别为（12.26±0.55）％、（20.83±1.22）％和（28.66±0.43）％。未处理组与处理组间及各处理组间转染效率的差异有统计学意义（P＜0.05）。MTT结果表明腺病毒M1介导的体外杀伤效应TSA处理组比未处理组增强（P＜0.05）,TSA各处理组间的体外杀伤效应随TSA浓度的提高而增强（P＜0.05）。结论　低浓度的TSA可以增强腺病毒对K562细胞的感染及杀伤效率,可以提高以腺病毒为载体的血液肿瘤基因治疗的疗效。     

Antineoplastic action of 5-aza-2'-deoxycytidine and histone deacetylase inhibitor and their effect on the expression of retinoic acid receptor beta and estrogen receptor alpha genes in breast carcinoma cells

PURPOSE: During tumorigenesis several cancer-related genes can be silenced by aberrant methylation. In many cases these silenced genes can be reactivated by exposure to the DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5-AZA-CdR). Histone acetylation also plays a role in the control of expression of some genes. The aim of this study was to determine the antineoplastic activities of 5-AZA-CdR and trichostatin A (TSA), either administered alone or in combination. in MDA-MB-231 breast carcinoma cells. The effects of these drugs (alone and in combination) on the expression of the tumor suppressor gene, retinoic acid receptor (RAR beta) and of the estrogen receptor alpha gene (ER alpha), whose expression is lost in the cell line used in the study, were also investigated. METHODS: MDA-MB-231 cells were treated with 5-AZA-CdR and TSA and the antitumor activity of these drugs was determined by clonogenic assay. Total RNA was extracted from the treated cells and RT-PCR was used to determine the effect of the treatment on the expression of RAR beta and ER alpha. Methylation-sensitive PCR analysis was used to confirm that lack of expression of both genes was due to hypermethylation of their promoter regions. A single nucleotide primer extension assay was also used to quantify the reduction in DNA methylation following drug treatment. RESULTS: Both 5-AZA-CdR and TSA alone showed significant antineoplastic activity. The combination of the two drugs was synergistic with respect to MDA-MB-231 cell kill. 5-AZA-CdR alone weakly activated the expression of both RAR beta and ER alpha. TSA alone only activated RAR beta, but not ER alpha. The combination of these agents appeared to produce a greater activation of both genes. CONCLUSIONS: The interesting interaction between 5-AZA-CdR and TSA in both cell kill and cancer-related gene reactivation provides a rationale for the use of inhibitors of DNA methylation and histone deacetylation in combination for the chemotherapy of breast cancer.     

曲古菌素A对HeLa细胞增殖及其基因表达的影响研究

Objective:To investigate the expressions of p53,RB1,Fas,c-fos,Ras,EGFR mRNA in human cervical cancer (HeLa) cell in response to the trichostatin A (TSA).Methods:We took count of HeLa cells in different incubation times with TSA (0.2 IJm/L).The result indicated that HeLa cells changed evidently when HeLa cells were incubated for 36 h.Then,we investigated the genes expression (mRNA levels) of HeLa cells after treatment for 36 h using SYBR green real-time PCR.Results:We demonstrated that trichostatin A (TSA) could make human cervical cancer (HeLa) cell morphological change and induce HeLa cell apoptosis.Furthermore,the data suggest that TSA-induced down-regulation of p53,RB1,Fas,but upregulated c-fos gene expression after treatment for 36 h,and Ras,EGFR did not show obvious response to TSA treatments.Conclusion:TSA has different effects on gene expression.     

Cell growth inhibition and gene expression induced by the histone deacetylase inhibitor, trichostatin A, on human hepatoma cells

OBJECTIVE: Histone deacetylase (HDAC) inhibitors have been reported to induce cell growth arrest, apoptosis and differentiation in tumor cells. The effect of the HDAC inhibitor, trichostatin A (TSA), on hepatoma cells, however, has not been well studied. In this study, we examined cell viability and gene expression profile in hepatoma cell lines treated with TSA. METHODS: To study cell growth inhibition and induction of apoptosis by TSA on human hepatoma cell lines including HuH7, Hep3B, HepG2, and PLC/PRF/5, cells were treated with TSA at various concentrations and analyzed by the 3-(4, 5-dimethyl-2-thiazolyl)-2H-tetrazolium bromide (MTT) and TUNEL assays, respectively. Changes in gene expression profile after exposure to TSA were assessed using a cDNA microarray consisting of 557 distinct cDNA of cancer-related genes. The levels of acetylated histones were examined by the chromatin immunoprecipitation (ChIP) assay using anti-acetylated histone H3 or H4 antibody. RESULTS: The MTT assay demonstrated that TSA showed cell growth inhibition not only in a concentration-dependent but also a time-dependent manner on all cell lines studied. The TUNEL assay also revealed the potential of TSA to induce apoptosis. The microarray analysis revealed that 8 genes including collagen type 1, alpha2 (COL1A2), insulin-like growth factor binding protein 2 (IGFBP2), integrin, alpha7 (ITGA7), basigin (BSG), quiescin Q6 (QSCN6), superoxide dismutase 3, extracellular (SOD3), nerve growth factor receptor (NGFR), and p53-induced protein (PIG11) exhibited substantial induction (ratio >2.0) after TSA treatment in multiple cell lines. ChIP assay, in general, showed a good correlation between the expression level of mRNA and levels of acetylated histones in these upregulated genes. CONCLUSIONS: This study showed cell growth inhibition and the gene expression profile in hepatoma cell lines exposed to TSA. The alteration in levels of acetylated histones was closely associated with expression of specific cancer-related genes in hepatoma cells.     

MAGE-A1 和MAGE-A3在脑胶质瘤组织中的表达及其临床意义

[摘要] 目的：探讨黑色素瘤抗原基因-A1（MAGE-A1）和MAGE-A3 在脑胶质瘤组织中的表达及其临床意义。方法：选取2006 年1 月至2010 年1 月河北医科大学第四医院神经外科手术切除的78 例脑胶质瘤组织标本和15 例车祸死亡捐献者的正常脑组织标本。用RT-PCR法检测胶质瘤组织中MAGE-A1 和MAGE-A3 的表达,分析其表达水平与患者预后的关系;用MSP-PCR术检测MAGE-A1 和MAGE-A3 基因启动子区的甲基化状态,分析两者表达与甲基化之间的关系;用RT-PCR法检测胶质瘤U251 和U87 细胞中MAGE-A1 和MAGE-A3 表达以及经DNA甲基转移酶抑制剂5-氮杂-2''-脱氧胞苷（5-Aza-CdR）和组蛋白去乙酰化酶抑制剂曲古抑菌素A（TSA）联合作用后两者的表达水平。结果：78 例胶质瘤组织中MAGE-A1 和MAGE-A3 mRNA阳性表达率分别为65.34%和38.46%,而在15 例正常脑组织中未发现2 种mRNA表达。MAGE-A1 阳性组患者5 年生存率较阴性组低（P<0.05）。MAGE-A1 和MAGE-A3 基因启动子区去甲基化水平与其在mRNA水平上的表达均有显著的相关性（均P<0.01）。未经5-Aza-CdR 和TSA处理的U87 细胞未见MAGE-A1 和MAGE-A3 mRNA的表达,U251 细胞则有少量表达。单独给予TSA不能引起MAGE-A1 和MAGE-A3 基因的表达激活,单独给予5-Aza-CdR 或与TSA合用可以引起该2 个基因的表达激活,且两者合用的作用优于单独给药。结论：脑胶质瘤组织中有不同程度的MAGE-A1 和MAGE-A3 基因表达,MAGE-A1 基因表达与患者的预后不良相关。DNA启动子区甲基化和组蛋白乙酰化是MAGE-A1和MAGE-A3基因表达激活的重要机制。     

Effect of Curcumin in Comparison with Trichostatin A on the Reactivation of Estrogen Receptor Alpha gene Expression,Cell Growth Inhibition and Apoptosis Induction in Hepatocellular Carcinoma Hepa 1-6 Cell lLine

Background: A multistep process with an accumulation of epigenetic alterations of tumor suppressor genes (TSGs) can induce cancer. Abnormal regional hypermethylation and histone deacetylation of several TSGs has been observed in hepatocellular carcinoma (HCC). Acetylation and deacetylation of histone are carried out by histone acetyltransferase (HAT) and histone deacetylase (HDAC) respectively. Besides, DNA methylation is carried out by DNA methyltransferases (DNMTs). Previously, we evaluated the effect of DNA demethylating agents and histone deacetylase inhibitors on HCC and colon cancer. This study aimed to evaluate the effect of curcumin (CUR) in comparison with trichostatin A (TSA) on estrogen receptor alpha (ERα) reactivation, apoptotic induction, and cell growth inhibition in HCC. Methods: the cells were cultured and treated with various concentrations of CUR and TSA and the MTT assay, flow cytometry assay and Real-Time RT-PCR were achieved to determine cell viability, cell apoptosis, and ERα gene expression respectively. Results: CUR indicated dose and time-dependent antiproliferative effects (P < 0.035). A similar antiproliferative effect was observed by TSA (P < 0.001). Both compounds indicated significant apoptotic effects in all different periods (P < 0.001), CUR indicated a more significant apoptotic effect than TSA (P < 0.001). The ERα gene expression quantity was increased significantly by treatment with CUR and TSA (P <0.012). Conclusion: CUR and TSA play important roles in restoring the ERα resulting in cell growth inhibition and apoptosis induction. Therefore, ERα may be a potential target for therapeutic intervention in the treatment of HCC.     

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9-cis-retinoic acid resistant (MKN-7 and Ho-1-N-1) and IFN-beta resistant cell lines (MKN-7, -28 and -45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP-ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21(WAF1), CREB-binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F-1, E2F-4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell-to-cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis-regulating proteins.