人胚肺成纤维细胞复制性衰老的基因表达变化

目的检测人胚肺成纤维细胞(2BS细胞)复制性衰老过程中的基因表达变化,并试图寻找细胞衰老的标志性基因。方法利用cDNA芯片对比检测28代龄和64代龄的2BS细胞基因表达谱,通过RT-PCR对挑选的9个差异基因在7个不同代龄2BS细胞中的表达进行验证分析。结果和年轻细胞相比,衰老2BS细胞有117种基因的变化幅度在2倍以上,46种基因在2.5倍以上;细胞衰老过程中下调的基因主要是细胞周期相关基因,上调基因主要参与细胞应激反应和蛋白分泌等过程;9个差异基因的RT-PCR验证结果与芯片结果基本一致。结论基因芯片是研究细胞衰老基因表达和调控网络的重要工具,可用于衰老特征性基因的筛选。     

应用抑制性消减杂交方法克隆人肝细胞癌差异表达基因

Diatchenko等[1]报道了一种新的快速克隆差异表达基因cDNA的方法,称为抑制性消减杂交(SSH),十分适用于克隆造成某种特殊细胞表型的特异表达基因,例如正常和病变之间的基因表达差异分析。人肝细胞癌(HCC)是我国高发的恶性肿瘤之一,其发生发展与多种基因的突变与表达异常有关。我们应用抑制性消减杂交方法克隆HCC与癌旁非癌组织之间差异     

人衰老二倍体成纤维细胞对烷化剂损伤的应答

目的　观察衰老的人胚肺二倍体成纤维细胞 (2BS)对烷化剂甲磺酸甲酯 (MMS)诱导的DNA损伤的应答。　方法　以体外培养的不同代龄的人胚肺 2BS为对象 ,以MMS诱导DNA损伤 ,以年轻细胞 (<30代 )为对照 ,观察衰老细胞 (>5 5代 )经MMS处理后的细胞形态、增殖特性、细胞周期的改变 ,并分别检测 gadd4 5、p2 1和p5 3等基因转录水平的表达变化 ,同时以非程序性DNA合成(UDS)和单细胞凝胶电泳试验测定DNA修复能力。　结果　经MMS诱导DNA损伤后 ,衰老细胞的细胞形态、生长曲线和细胞周期的变化均不及年轻细胞明显 ;gadd4 5、p2 1和 p5 3等基因的可诱导性表达均低于年轻细胞 ;同时 ,衰老细胞总的及单个细胞的修复能力较年轻细胞明显下降。　结论　衰老 2BS细胞对MMS诱导的DNA损伤后的细胞应答变化能力下降 ,且其修复能力的减退可能与基因的可诱导性表达下降有关。     

日本血吸虫病肝纤维化小鼠肝星状细胞差异表达cDNA文库的构建和筛选

目的构建日本血吸虫病肝纤维化小鼠肝星状细胞差异表达基因的消减cDNA文库,并筛选差异表达基因。方法取日本血吸虫尾蚴经腹部感染小鼠致肝纤维化。采用抑制性消减杂交技术(SSH),分离小鼠肝星状细胞(HSC)及正常小鼠HSC,通过对比寻找差异表达基因。将其与T载体连接(T/A克隆),其产物转化大肠埃希菌DH5α,经文库扩增后,随机挑取白色克隆进行酶切鉴定,克隆经过正、反向杂交,阳性克隆经过测序和BLAST(局部相似性基本查询工具)进行表达序列标签(ESTs)差异基因分析。最后经模拟Northern印迹确认基因表达差异。结果扩增消减cDNA文库获得400余个白色阳性克隆,随机挑取的克隆经酶切鉴定后均有200～600 bp插入片段。ESTs分析获得76个序列,其中70个序列提示与血吸虫病肝纤维化或与其相关的基因,6个在公共数据库中未找到同源序列片段。结论用SSH法及T/A克隆技术成功构建了肝纤维化小鼠HSC与正常小鼠HSC差异表达基因的消减cDNA文库。     

肝硬化患者发生的肝细胞癌差异表达基因文库的构建及初步筛选

目的 与肝硬化对比，研究肝细胞癌中差异表达的基因，构建在肝硬化基础上发生癌变的肝细胞之差异表达的cDNA消减文库。方法 采用抑制性消减杂交获得差异表达基因片段，T/A克隆，蓝白斑筛选，在自动测序仪上进行基因测序，做同源性表达序列标签长度在112-755bp之间，序列分析揭示28个克隆是来自以前描述过的基因，而另外7个在GenBank/EMBL/DDBJ数据库没有匹配序列，可能代表新基因。结论 实验结果表明从肝硬化发展至肝细胞癌涉及到多个基因的差异表达；本研究在成功地构建出流水作消减基因文库的同时，也证明了抑制性消减杂交技术对寻找差异表达基因是十分适用的。     

抑制性消减杂交技术原理及应用

0 引言抑制性消减杂交(suppression subtractive hybridization, SSH)技术是一种鉴定、分离组织细胞中选择性表达基因的技术。其原理是以抑制性多聚酶链反应(PCR)反应为基础的cDNA消减杂交技术。通过合成两个不同的接头,连接于测试cDNA片段的5’末端,达到选择性扩增差异性表达的cDNA片段,抑制非目的cDNA的扩增。该技术与其他消减杂交技术相比具有假阳性率低、敏感性高、效率高等优点而得到广泛应用, 1 抑制性消减杂交技术产生的背景高等真核生物细胞中约含有4-10万个不同的基因,但在生物体的发育过程中只有15％的基因得以表达。这     

筛选鉴定不稳定性心绞痛淋巴细胞差异表达基因

目的　筛选鉴定不稳定性心绞痛淋巴细胞差异表达基因 ,从分子水平探讨不稳定性心绞痛的发病机理。为不稳定性心绞痛早期诊断和潜在可能的基因治疗提供线索。方法　应用抑制性消减杂交技术筛选不稳定性心绞痛淋巴细胞和稳定性心绞痛淋巴细胞差异表达基因。将获得的顺向或逆向消减产物与 pGEM TEasyVector连接并转化大肠杆菌JM10 9,采用蓝白斑筛选和菌落PCR筛选有插入片段的克隆。将获得的菌落PCR产物分别等量点在 2张Hybond N 膜的同一位置上 ,同时与顺向消减和逆向消减产物进行斑点杂交 ,进一步筛选差异表达的cDNA片段。以获得的序列表达标签 (EST)为探针 ,与顺向消减和逆向消减产物进行反向Northern杂交 ,将获得的阳性EST测序 ,并进行同源性比较。结果　斑点杂交结果显示 :在不稳定性心绞痛组获得 93个阳性克隆 ,在稳定性心绞痛组获得 112个阳性克隆 ;反向Northern杂交结果显示 :在不稳定性心绞痛组获得 12个阳性EST ,在稳定性心绞痛组获得 2 0个阳性EST ,在各组中随机选取 5个阳性EST测序并进行同源性比较 ,全部为已知基因的部分序列。结论　这些EST与不稳定性心绞痛的发生与发展密切相关     

日本血吸虫消减雌性成虫cDNA文库的建立及其特异表达基因的筛选

目的 筛选雌性日本血吸虫特异表达基因。方法 感染日本血吸虫6周的家兔,用静脉灌注法收集成虫,经核糖核酸固定液固定,分别提取雌、雄成虫总RNA,纯化后获得mRNA,并反转录为cDNA。用抑制性消减杂交技术(SSH)构建雌、雄成虫正向消减(雌虫消减雄虫)及反向消减(雄虫消减雌虫)cDNA文库。用斑点杂交法筛选差异表达基因,挑选目标基因片段(与正向消减探针杂交的信号明显高于与反向消减探针杂交信号的克隆)进行测序、同源性搜索及基因功能预测分析。以日本血吸虫肌动蛋白(actin)基因作内参照,用半定量PCR(semi-quantitative PCR)鉴定目标基因在雌、雄虫体内的表达。结果 得到正向消减及反向消减cDNA文库,斑点杂交筛选出50个雌虫特异性表达的克隆,经测序得到42个表达序列标签(EST),其中,有17个基因(占40.5%)与已知日本血吸虫卵壳蛋白基因高度同源;17个基因(占40.5%)与日本血吸虫未知基因高度同源、且有一小片段与卵壳蛋白基因高度同源;有8个基因(占19.0%)与日本血吸虫其他未知基因高度同源。半定量PCR结果,6个基因在雌虫体内的表达水平明显高于雄虫,分别与GenBank的血吸虫卵壳蛋白基因AY222885、AY222895、AB017097、AF519182、M32281及血吸虫其他基因AY813556高度同源。结论 构建了雌、雄成虫正向消减及反向消减cDNA文库。用SSH可筛选日本血吸虫雌性特异性表达基因。     

丙型肝炎病毒非结构蛋白NS3反式激活基因2的克隆化研究

目的 应用抑制性消减杂交技术(SSH)及生物信息学技术(bioinformcs)筛选并克隆丙型肝炎病毒(HCV)非结构蛋白3(NS3)反式激活新型靶基因，进一步阐明HCV感染相关疾病的发病机制。方法 以HCVNS3蛋白表达质粒pcDNA3．1(-)-NS3转染HepG2细胞，以空载体pcDNA3．1(-)为平行对照，提取mRNA并进行抑制性消减杂交分析。并应用分子生物学技术，结合生物信息学技术，克隆HCvNS3反式激活作用的新的靶基因。结果 对于所获基因片段序列分析表明，其中之一为新型基因片段，从HepG2细胞提取总RNA，以逆转录多聚酶链反应(RT-PCR)技术扩增获得该新基因的全长序列，并测序证实，因其可以被NS3蛋白反式激活，故命名为NS3TP2，在GenBank中注册，注册号为AYll6970。NS3TP2基因的编码序列全长为1299个核苷酸(nt)，编码产物由432个氨基酸残基(aa)组成。结论 HCVNS3是一种典型的病毒基因组编码的具有反式激活作用的蛋白，而抑制性消减杂交(SSH)是一种鉴定、分离组织细胞中选择性表达基因的技术。通过这种技术，发现了HCVNS3反式激活作用的新的靶基因，这一发现，为进一步研究HCVNS3蛋白反式激活作用的分子生物学机制和探索新型治疗技术奠定基础。     

抑制性消减杂交技术筛选γ-氨基丁酸调节的肝星状细胞系靶基因

目的:应用抑制性消减杂交技术(SSH)筛选γ-氨基丁酸(GABA)作用肝星状细胞系HSC-T6后的差异表达基因.方法:10 umol/L的GABA作用于HSC-T6细胞24 h,提取mRNA,用分光光度计进行定量分析.以GABA处理和未处理的T6细胞mRNA为模板逆转录合成双链cDNA(dscDNA),并分别标记为Tester和Drivet,酶切后与接头连接,经两次杂交,构建消减杂交文库,将消减文库的扩增产物进行转化、克隆分析后应用生物信息学技术将测得序列再进行同源性分析.结果:15种基因表达上调,其中包括与DNA合成、线粒体、肿瘤抑制以及凋亡相关的4类基因,结果显示GABA可能促进HSC-T6细胞增殖而抑制凋亡.结论:用SSH技术可以成功获得GABA刺激肝星状细胞基因上调的监测,证明GABA能够影响肝星状细胞的基因表达谱.     

Expression profiles of subtracted mRNAs during cellular senescence in human mesenchymal stem cells derived from bone marrow

Cellular senescence is an irreversible cell cycle arrest that limits the replicative lifespan of cells. Senescence suppresses development of tumors by regulating aging factors, such as cyclin dependent kinase inhibitor (CKI) and telomerase. Suppression subtractive hybridization (SSH) was used to identify genes that were differentially expressed between young human mesenchymal stem cells (Y-hMSCs) and senescent human mesenchymal stem cells (S-hMSCs). We selected positive clones that were functionally characterized by referring to public databases using NCBI BLAST tool. This search revealed that 19 genes were downregulated, and 43 genes were upregulated in S-hMSCs relative to Y-hMSCs. Among subtracted clones in Y-hMSCs, most of genes markedly were related to metabolic functions. These genes, PDIA3, WDR1, FSTL1, COPG1, LMAN1, and PDIA6, significantly downregulated. Conversely, genes for subtracted clones in S-hMSCs were mostly associated with cell adhesion. In particular, the expression levels of 9 genes, HSP90B1, EID1, ATP2B4, DDAH1, PRNP, RAB1A, PGS5, TM4SF1 and SSR3, gradually increased during senescence. These genes have not previously been identified as being related to cellular senescence, but they seemed to be potentially affected during cellular senescence.     

细胞周期抑制因子p16可诱导人成纤维细胞发生衰老样变化

目的 探讨细胞周期抑制因子p16在细胞衰老中的作用。方法 利用逆转录病毒载体将p16基因转染入人胚肺二倍体成纤维细胞2BS中,获得高表达,检测其对2BS细胞衰老的影响。结果 与对照组细胞相比,p16基因转染后,2BS细胞生长速度下降了约50％,细胞周期阻滞于G1期,细胞对生长因子刺激的反应性下降了79．4％,细胞形态呈衰老细胞样变化。结论 p16在人二倍体成纤维细胞的衰老过程中起促进作用。     

Subtractive hybridization of mRNA from early passage and senescent endothelial cells

Regulation of cellular processes that eventually lead to a state of growth arrest is an important manifestation of in vitro cellular senescence caused and accompanied by variations of the gene expression pattern. Whereas these changes at the mRNA level have been studied mainly in fibroblast cultures, we concentrated on endothelial cells that represent an accepted model for vascular systems and may be involved in the pathogenesis of diseases related to aging. To isolate differentially expressed genes, we created a subtractive cDNA library using mRNA from senescent (35 passages) and young (five passages) human umbilical vein endothelial cells (HUVECs). Candidate clones were isolated from the cDNA library, differential expression was confirmed by Northern blot analyses and sequences were compared with a genbank data base. Because many mRNAs were below the detection limit of Northern blot analysis, we were forced to establish a more sensitive PCR based method (ATAC-PCR) to quantify and confirm altered levels of gene expression. Several mRNAs were found to be upregulated in senescent HUVECs including two components of the extracellular matrix (ECM): plasminogen activator inhibitor and fibronectin. Elevated expression of both has already been described in senescent cells. The mRNAs of TGF-beta-inducible gene H3 (beta-IG-H3; ECM protein), insulin-like growth factor binding protein (IGFBP-3), p53-inducible gene (PIG3) a protein involved in vesicular transport (SEC13R) and ribosomal protein L28 have likewise been shown to be preferentially expressed in senescent cells. Because studies support the involvement of ECM components, TGF-beta and p53 in tumor suppressing mechanisms, our data supports the hypothesis that cellular senescence and upregulation of ECM proteins may be associated with tumor preventive functions.     

Induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cells

5-Bromodeoxyuridine (BrdU) universally induces a senescence-like phenomenon in mammalian cells. To assess this phenomenon at the level of gene expression, we constructed a PCR-based subtractive cDNA library enriched for mRNA species that immediately increase by administration of BrdU to HeLa cells. Candidate cDNA clones were isolated by differential colony hybridization, and then positive clones were identified by Northern blot analysis. Sequencing analysis revealed that the identified cDNA species were classified into three groups: widely used senescence-markers, known species whose relevance to senescence is yet to be reported, and known or novel ESTs. As expected, the majority of them showed an increase in expression in senescent human diploid fibroblasts. These results suggest that similar mechanisms operate in the regulation of BrdU-induced genes and senescence-associated genes.     

Further studies on the genetic and biochemical basis of cellular senescence

Cell fusion analysis, exploiting the fact that the phenotype of immortality is recessive in hybrids, has allowed the assignment of 26 different immortal human cell lines to at least four complementation groups for indefinite division. This indicates that there are at least four sets of genes or processes involved in the mechanisms leading to cellular senescence. We have also observed alterations in gene expression accompanying senescence that induce the the expression of a protein inhibitor of DNA synthesis, expression of new cell surface epitopes as identified by monoclonal antibodies specific to senescent cells, and changes in the extracellular matrix. We have yet to determine whether these changes in gene expression are casual or the result of senescence. The assignment of immortal cell lines to specific complementation groups now allow for a focused approach to identify the normal growth regulatory genes that have been modified to yield immortal cells and determine whether certain senescent cell specific patterns of gene expression continue to be expressed in immortal cells within a group. In addition, the isolation of senescent cell-specific antibodies provides for the first time the tools with which to probe the relationship between in vitro and in vivo aging.