非何杰金淋巴瘤p15基因操纵区5’-CpG岛的甲基化的研究

目的：探索p15基因异常甲基化在非何杰金淋巴瘤(NHL)中的变化及其在各分型中的表达。 方法：用特异PCR（MSP）的方法检测32例非何杰金淋巴瘤石蜡标本p15基因甲基化；检测非何杰金淋巴瘤患者标本21例，用RT-PCR方法测定p15基因的表达。 结果:非何杰金淋巴瘤p15基因操纵区甲基化的发生率为18.9%（10/53），高度恶性比低度恶性更易发生甲基化，其发生率分别为27.3%和0%。 结论:p15基因甲基化可能是非何杰金淋巴瘤发病的原因之一，并与病程进展有关。     

Methylation status of immunoglobulin x e segments correlates with their recombination potential

We have previously shown that unlike endogenous ? genes, unrearranged ? transgenes undergo V?-J? recombination in T as well as B cells of transgenic mice. To determine whether the difference in recombination specificity of the transgenic and endogenous ? genes is associated with differences in DNA structure, the methylation status of the endogenous genes and three unrearranged ? transgenes was compared. The J?-C? locus of the transgenes was found to be hypomethylated in all tissues of the transgenic mice. In contrast, methylation of the endogenous ? genes was tissue and developmentally regulated. Hypomethylation of the endogenous J?-C? region occurs only in cells of the B lineage undergoing, or having completed ? gene recombination. Transfection of fibroblasts from transgenic and control mice with the recombination activating genes, Rag1 and Rag2, led to a high level of rearrangement of the hypomethylated transgenic, but not the endogenous ? genes. These results suggest that hypomethylation defines an accessible state of the ? locus and that methylation/demethylation could be involved in the control of ? gene rearrangement during lymphocyte differentiation.     

软组织平滑肌肉瘤中p16基因的甲基化检测

目的 探讨软组织平滑肌肉瘤（ＬＭＳ）中p16基因ＩＮＫ４Ａ的甲基化状态及其与p16表达的关系。方法 应用ＭＳＰ法检测３８例软组织平滑肌肉瘤,１０例平滑肌瘤及５例正常平滑肌组织中p16基因ＩＮＫ４Ａ的甲基化状态,用免疫组织化学ＳＰ方法检测p16蛋白表达情况。结果 ３８例ＬＭＳ中９例发生异常甲基化,异常甲基化率为２３.7%（９／３８）。其中,７例p16蛋白表达阴性,２例p16蛋白弱阳性,在p16蛋白表达阴性的ＬＭＳ中,异常甲基化率为５０％（７／１４）。结论 p16基因第一外显子启动子区５‘CpG岛的异常甲基化是导致p16基因失活、蛋白缺如的重要基因外机制,并可能参与肿瘤的发生。     

HIV-1感染引起组蛋白乙酰化和增加p21WAF1表达

目的：DNA甲基化和组蛋白乙酰化是基因表达调控的主要形式。人类免疫缺陷病毒（HIV-1）可引起T淋巴细胞DNA甲基化酶上调。本文旨在明确HIV-1对细胞周期依赖刺激酶抑制剂p21^WAF1表达的影响。方法：建立HIV-1感染的Hut78细胞系；以RT-PCR和Westem blotting 分析p21^WAF1表达情况；以亚硫酸氢钠修饰DNA和基因测序，研究p21^WAF1基因启动子甲基化，以Western blot-ting 和染色体免疫测定探究总组蛋白和与p21^WAF1基因启动子相关的组蛋白乙酰化水平。并以GST pull-down和免疫沉淀分析HIV-1导致乙酰化及乙酰化引起p21^WAF1过表达的可能机理。结果：HIV-1感染后，其反式激活蛋白Tat与辅助转录因子P/CAF、hGCN5结合，共同刺激组蛋白H3乙酰化。尽管p21^WAF1启动子部分区域有甲基化发生，但p21^WAF1表达仍上调。这可能与E2A对p21^WAF1的作用有关。结论：HIV-1感染可引起T淋巴细胞p21^WAF1基因的甲基化和乙酰化紊乱，导致p21^WAF1表达增强。     

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl₂ protoplast fusion protocol. Hygromycin B-resistant (HygB^R) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygB^R progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.     

3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性

目的对3株肺癌细胞中9个肿瘤相关基因的启动子甲基化状态和部分基因表达的相关性进行评估.方法通过甲基化特异性多聚酶链反应法(MSP)结合DNA测序来确定3株肺腺癌细胞株(A549,SH77和SPE-A-1)中9个基因的启动子CpG岛的甲基化状态:APC,CDH13,DAPK1,MGMT,MYOD1,p16INK4a,p73,RAR-β和WT1基因.同时采用半定量RT-PCR的方法来量度下列5个基因的表达:CDH13,MGMT,MYOD1,RAR-β和WT1基因.结果在9个受检基因之中,下列4个基因在三株细胞中均呈同样的甲基化谱式:仅有去甲基化:APC和DAPK1,以及兼有甲基化和去甲基化:p73和p16INK4a.而其余5个基因则表现为不同的甲基化模式.尽管,这5个基因的表达谱式大体上符合其启动子CpG岛甲基化的表达静息的原则,个别的例外亦是有的.这提示着与DNA甲基化状态无关的机制可能也会参与该基因的表达调控.结论肺癌细胞株中的肿瘤相关基因的启动子CpG岛的过甲基化可与该基因的表达状态有较高的相关性.     

检测痰脱落细胞p16基因甲基化对周围型肺癌的诊断价值

目的　探讨检测痰脱落细胞p16基因甲基化对周围型肺癌的诊断价值。方法　应用甲基化特异性PCR(methylation specificPCR ,MSP)对 5 0例周围型肺结节患者及 2 0例正常人痰脱落细胞p16基因甲基化改变进行检测 ,将检测结果与术后病理报告相对照。结果　周围型肺癌痰脱落细胞 p16MSP阳性率( 2 7/ 44 ,61.4%)明显高于良性周围型肺结节 ( 1/ 6,16.7%)及正常人 ( 3 / 2 0 ,15 .0 %) ( χ2 值分别为 4.2 81和11.869,P <0 .0 5 ) ,良性周围型肺结节 p16MSP阳性率与正常人无显著性差异 ( χ2 =0 .13 6,P >0 .0 5 )。鳞癌和腺癌患者痰脱落细胞 p16MSP阳性率无显著性差异 ( χ2 =3 .416,P >0 .0 5 )。如果以痰脱落细胞 p16MSP阳性作为判断肺结节恶性的标准 ,其诊断周围型肺癌的阳性预测值为 96.4%,阴性预测值 2 2 .7%,灵敏度 61.4%,特异度 83 .0 %。结论　检测痰脱落细胞 p16基因甲基化对周围型肺癌具有辅助诊断价值     

食管癌细胞系中MT-3基因CpG岛甲基化的意义

目的研究食管癌细胞系中MT-3基因CpG岛甲基化与其mRNA表达的关系。方法选取4种食管癌细胞系OE21，OE19，OE33和TE7，经重亚硫酸钠处理后，采用限制性酶切图谱分析(COBRA)和逆转录聚合酶链反应(RT-PCR)技术，在DNA甲基转移酶抑制剂5-氮2^ -脱氧胞苷(5-aza-CdR)处理前后，对其MT-3基因CpG岛的甲基化情况及mRNA的表达情况进行对比研究。结果4种食管癌细胞系的MT-3基因均存在不同程度的CpG岛超甲基化。经5-aza-CdR处理后，超甲基化状态解除，mRNA的表达水平也较处理前明显提高(P=0.002)。结论食管癌细胞系中MT-3基因CpG岛的超甲基化可抑制其mRNA表达。当其超甲基化解除后，MT-3基因的mRNA表达水平也会相应升高。