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细胞在增殖,分化及对外界刺激反应过程中,可伴随某些特殊基因的表达,利用这一特性,通过比较细胞在不同状态及不同分化阶段基因表达的差异,可以发现与细胞分化/生长相关的基因,近年来国外学者发展了几种能有效进行差异表达基因分析的技术和方法,包括差异显示PCR,代表性差异分析,抑制性消减杂交及DNA微阵列等。本文主要对目前主要技术方法作一综述。 相似文献
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玉米耐铝毒基因的分离 总被引:12,自引:0,他引:12
以抑制消减杂交(SSH)为手段,以玉米对铝敏感的自交系Mo17和耐铝的自交系TL94B为材料,分别构建它们的正向和反向消减文库,分别筛选获得了124、47、103和64个阳性克隆。对文库的鉴定表明,插入片段分布在0.25-1.0kb之间,阳性克隆率在18%左右。对338个阳性克隆进行测序,得到232种表达序列标签(EST),其中70.2%的EST可推测其功能。结果表明,玉米的铝离子胁迫反应涉及胁迫因子的信号传导、响应基因的转录表达与调控、物质的合成与运输、细胞结构和功能的改变等。 相似文献
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鉴别差异表达基因的新方法—抑制消减染交法(SSH) 总被引:3,自引:0,他引:3
抑制消减杂交法是最近报道的能有效鉴别两个不同细胞群差异表达基因的新方法。该方法是以抑制PCR和消减杂交法为基础,在一轮反应中实现mRNA的均等化和消减步骤。本文对SSH法的原理,优缺点作了介绍,并与其它检测差异表达基因的方法,如mRNA差别显示江,代表性序列差异分析比较,认为SSH方法具有高效快速,高敏感性和假阳性低等优点。 相似文献
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鉴别差异表达基因的新方法──抑制消减杂交法(SSH) 总被引:3,自引:0,他引:3
抑制消减杂交法(SuppressicSubtractiveHybridization,SSH)是最近(1996)报道的能有效鉴别两个不同细胞群差异表达基因的新方法[1,2]。该方法是以抑制PCR和消减杂交法为基础,在一轮反应中实现mRNA的均等化和消减步骤。本文对SSH法的原理、优缺点作了介绍,并与其它检测差异表达基因的方法,如mRNA差别显示法(DDRT-PCR)、代表性序列差异分析(RDA)比较,认为SSH方法具有高效快速、高敏感性和假阳性低等优点。同时,625个克隆中有很大一部分是转录物丰度很低的基因,40%的克隆可能为新的基因。因此,SSH可作为鉴别差异表达基因和克隆新基因的有效方法。 相似文献
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为克隆肺腺癌分化相关基因, 采用诱导分化与消减杂交相结合的策略, 建立了全反式维甲酸(RA)诱导前后人肺腺癌细胞系的cDNA消减文库, 得到124个cDNA消减克隆. 经加减法杂交差异筛选、DNA和RNA印迹、cDNA全序列测定和生物学功能分析, 分离到3个在人肺腺癌细胞系分化过程中由RA激活而特异表达的新的cDNA序列这一策略和技术路线适用于分离细胞中呈过量表达或表达抑制基因的cDNA克隆, 并具有反映细胞分化过程中基因表达动态变化特征和相对简便适用的特点. 相似文献
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大鼠心肌缺血预适应诱导表达上调基因的筛选和鉴定 总被引:7,自引:1,他引:7
采用反复短时间结扎及松解左冠状动脉前降支构建大鼠心肌缺血预适应动物模型,运用抑制消减杂交技术建立大鼠心肌缺血预适应诱导表达上调基因的消减cDNA文库,通过反向RNA点杂交对部分文库基因进行差异表达初筛,选取表达差异最明显的85个基因进行测序,获得了31个核编码基因和18个新基因(EST),核编码基因中有相当一部分与细胞保护或信号转导有关,新基因已被GenBank收录.从已测序基因中任选5个进行RT-PCR检测,并任选2个进行RNA印迹检测,均证实在缺血预适应时表达增高.上述结果为进一步深入研究缺血预适应心肌保护作用的分子机制和克隆新的缺血预适应相关基因提供了重要信息. 相似文献
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To isolate the over-expressed genes in human renal cell carcinoma (RCC) and analyze its molecular basis of carcinogenesis,
we used the mRNA from human RCC tissues as tester and that from the matched normal kidney tissues as driver to construct the
suppression subtractive hybridization library. 379 of the subtracted clones were arrayed onto a nylon membrane and the over-expressed
genes were then screened by hybridizing the filter with radioactively labeled cDNA from RCC and matched normal kidney tissues.
67 clones over-expressed in RCC by a factor of 6 or more were sequenced and its identities were analyzed in GenBank database.
4 clones were previously unknown fragments and 2 clones represent KIAA genes. The rest clones were the known genes and some
of them were RCC-related, including vascular endothelial growth factor, vimentin and tissue factor. Most of the known genes
were the RCC-related genes previously unknown, including zinc ribbon domain-containing 1 protein (ZNRD1), pituitary tumor transforming gene1 (PTTG1). Northern blot and semi-quantitative RT-PCR confirmed that the mRNA levels of the 3 novel fragments and 1 KIAA and 3 known
genes were significantly higher in RCC than in the matched normal kidney tissues. Immunohistochemical and Western blot analysis
for PTTG1 and ZNRD1 revealed increased protein level in RCC. The over-expressed genes in RCC are the potential molecular targets
for diagnosis and therapy and it is very important to understand the molecular mechanism of RCC through the profile of over-expressed
genes. 相似文献
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Rambow F Malek O Geffrotin C Leplat JJ Bouet S Piton G Hugot K Bevilacqua C Horak V Vincent-Naulleau S 《Pigment cell & melanoma research》2008,21(2):147-161
Partial and some few cases of complete spontaneous regression have been observed in cutaneous melanoma patients but little is known about the molecular mechanisms involved. The Melanoblastoma-bearing Libechov Minipig (MeLiM) is a suitable animal model to study the phenomenon of spontaneous regression because MeLiM pigs exhibit naturally occurring melanomas which regress completely 6 months after birth. In this study, we used suppression subtractive hybridization (SSH) to identify molecular determinants of melanoma regression within swine melanoma tissues and melanoma cell cultures. Several markers involved in cell-adhesion, -communication, -motility, signal transduction, negative regulation of cell proliferation, transport and immune response were identified that correlated with melanoma regression whereas the main genes involved in melanin synthesis showed a strong downregulation. For the most differentially expressed genes, we validated the results obtained by SSH with qRT-PCR and with immunohistochemistry for some of them (CD9, MITF, RARRES1). Most notable, for the first time in melanoma, we identified the retinoic acid responder 1 gene (RARRES1) as a main actor of the regression process in melanoma. This first gene expression study in swine melanoma regression, may contribute to the finding of new therapeutic targets for human melanoma treatment. 相似文献
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目的:探讨大鼠在血管钙化消退过程中基因表达的变化。方法:选取6周龄SPF级雄性SD大鼠24只,随机分成3组(n=8),分别为对照组、钙化组和消退组。钙化组和消退组制作血管钙化模型(vitamin D3 plus nieotine,VDN),对照组生理盐水和花生油灌胃。钙化组和对照组于实验第8周处死,消退组继续饲养至16周处死,测定各组大鼠动脉组织钙含量并作病理检查。采用抑制性消减杂交方法将消退组和钙化组大鼠血管cDNA作差减杂交,分离消退组较钙化组高表达或低表达基因的cDNA片段,建立消退组和钙化组的差异表达文库,扩增、鉴定文库并测序阳性克隆,BLAST比对测序序列。随机挑选4个基因进行RT-PCR验证和DNA条带半定量分析。结果:①血管组织钙含量测定显示钙化组((15.34±2.51)mg/g)较对照组((5.20±0.75)mg/g)血管组织钙含量明显升高(P〈0.01);消退组((12.73±1.89)mg/g)较钙化组降低(P〈0.05);②构建了血管钙化的差减文库,对所获阳性克隆进行测序和BLAST比对分析,获得28个表达上调基因和22个表达下调基因。RT-PCR验证示Prdx3,Ank2,Ror2,Abcel等基因在消退组和钙化组间差异表达,消退组较钙化组表达增高,平均约为后者的1.7倍。结论:VDN模型诱导的大鼠血管钙化可以发生主动消退。钙化消退过程中焦磷酸合成相关基因、谷氨酸信号相关基因、还原及凋亡调节基因表达上调,同时见较多骨化相关基因及氧化活性等基因表达下调。钙化抑制基因的表达增多而钙化促进基因表达的下降可能是钙化发生主动消退的内源性机制。 相似文献
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生命活动的各个进程伴随着不同基因的选择性开启和关闭。如何有效地分离克隆各种差异表达的基因,成为分子生物学研究的一个努力方向,大量差异基因分离策略因之问世。本文扼要介绍了近年来发展的几种主要分离策略,并详细介绍一种新的差异基因分离方法--抑制差减杂交。 相似文献
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AI Junkui ZHANG Zhiwen XIN Dianqi ZHU Hongjian YAN Quanjian XIN Zhongcheng NA Yanqun & GUO Yinglu . Institute of Urology Peking University & Department of Urology First Hospital Peking University Beijing China . Department of Physiology Peking University Health Science Center Beijing China . Department of Urology Xijing Hospital the th Military Medical University Xi抋n China 《中国科学:生命科学英文版》2004,47(2):148-157
Renal cell carcinoma (RCC) is a common uro- genital malignancy and often shows odd biological features. RCC accounts for approximately 2% of ma- lignancies worldwide. The incidence of and mortality from RCC have continuously increased during the last 50 years. One third of the patients already have me- tastases when first consulting the doctors. Another 30%—40% of patients develop metastasis after surgi- Identification of over-expressed genes in human RCC 149 cal excision of the pri… 相似文献
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抑制消减杂交和辐射小鼠血虚模型筛选"四物汤"诱导表达的基因 总被引:7,自引:0,他引:7
应用抑制性消减杂交技术构建受辐射小鼠血虚模型在中药四物汤诱导前后消减cDNA库,并从中克隆鉴定出与四物汤药效相关基因。从受辐射小鼠四物汤诱导前后骨髓细胞中提取mRNA并合成cDNA,分别作为tester和driver进行消减杂交及抑制性PCR,将PCR产物与载体连接构建cDNA库,高效电转化大肠杆菌进行库扩增,随机挑取其中的克隆进行酶切、测序分析。成功构建了具有高消减效率的受辐射小鼠四物汤诱导前后的消减cDNA库。库挑选得到512个阳性克隆。随机挑取30个插入片段测序,生物信息学分析结果显示大部分与红细胞分化、骨髓组织修复、结构重建有密切关系,其中8个为新基因片段。应用抑制性消减杂交技术所构建的受辐射小鼠四物汤诱导前后cDNA消减库为大批量筛选、克隆四物汤药效特异性相关基因奠定了基础。并从分子水平上发现四物汤对抑制凋亡、促进骨髓组织修复和结构重建、诱导红细胞分化的基因具有调节作用,可能和四物汤补血作用有关。 相似文献
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Identification of differentially expressed genes like cofilin2 in growing collateral arteries 总被引:1,自引:0,他引:1
Boengler K Pipp F Broich K Fernandez B Schaper W Deindl E 《Biochemical and biophysical research communications》2003,300(3):751-756
Arteriogenesis, the growth of pre-existing collateral arteries, can be induced in rabbits by occlusion of the femoral artery. In order to analyze the differential gene expression in arteriogenesis, cDNA of collateral arteries 24h after femoral occlusion or sham operation was subjected to suppression subtractive hybridization (SSH). We demonstrated an upregulation of the U6 snRNA binding protein Lsm5, cytochrome b, an expressed sequence tag, and the actin-depolymerizing factor cofilin2 mRNA in collateral arteries 24h after femoral ligation. For cofilin2, we also detected an increase in the protein level and a localization predominantly in smooth muscle cells of collaterals. Simultaneously with the upregulation of cofilin2 we found a downregulation of the alpha-smooth muscle actin mRNA in growing collateral arteries. In summary, our data showed an augmented expression level of genes contributing to different fundamental processes of arteriogenesis. 相似文献