肾癌组织消减文库的构建与肾癌特异表达基因克隆

目的 构建人肾癌组织与正常肾组织差异表达的cDNA消减文库,从文中克隆鉴定出肾癌特异性表达的基因奠定基础。方法 应用抑制性消减杂交技术,分别从肾癌及正常肾组织中提取poly(A) RNA;依次合成单链及双链cDNA,分别与2种不同的接头衔接,再与正常肾cDNA进行2次消减杂交及2次抑制性PCR;将产物T／A载体连接接构建成功cDNA消减文库。结果 构成功具有高消减效率的人肾癌组织cDNA消减文库,文库扩增后得到350个阳性克隆,其中95％克隆均含50－400bp插入片段。结论 应用抑制性消减杂交技术所构建的人肾癌组织cDNA消减文库为进一步大批量筛选、克隆肾癌特异性表达的基因奠定了基础。     

应用抑制性消减杂交方法构建斑秃毛乳头差异表达基因文库

目的：构建斑秃区与正常毛囊毛乳头细胞（DPC）差异表达的cDNA正向和反向消减杂交文库，为从中克隆鉴定出斑秃特异性表达和生长期DPC特异性表达的基因奠定基础。方法：应用抑制性消减杂交技术，分别从斑秃区DPC及正常头皮DPC提取总mRNA；依次合成单链及双链cDNA，分别与2种不同的接头连接，再进行正向和反向的2次消减杂交及2次抑制性PCR，将产物与T／A载体连接构建cDNA消减文库。结论：构建成功具有高消减效率的斑秃区及正常头皮DPC cDNA消减文库，文库扩增后得到120个阳性克隆，其中90个克隆含有100－500bp插入片段，结论：应用抑制性消减杂交技术所构建的斑秃区及正常头皮DPC cDNA消减文库，为进一步批量筛选，克隆斑秃区及正常头皮DPC特异性表达的基因奠定了基础。     

肾癌抑制性消减杂交文库的构建及意义

目的　应用抑制性消减杂交技术构建人肾癌组织与正常肾组织间差异表达的cDNA组成的消减文库。　方法　分别从肾癌组织和正常肾组织提取polyA RNA ,合成双链cDNA ,经RsaI酶切后将肾癌cDNA分为两组并加上不同的DNA接头 ,再与过量正常肾组织cDNA进行两次消减杂交及两次抑制性PCR ,PCR产物与T/A载体连接并转化大肠杆菌构建成cDNA消减文库 ,文库扩增后随机挑取克隆进行酶切、测序及同源性分析。　结果　文库共包含 4 14个阳性克隆 ,随机挑取 2 6 5个阳性克隆提取质粒并酶切分析 ,其中 2 4 6个克隆有插入片段。将其中 4 0个克隆进行测序 ,表明 1个克隆为新基因片段 ,其余 39个源于 35个已知基因。　结论　该消减杂交文库质量可靠 ,它的成功构建为进一步筛选、克隆肾癌差异表达基因提供了依据     

应用抑制性消减杂交技术克隆肾癌转移相关基因

目的:应用抑制性消减杂交技术构建人高侵袭性肾癌细胞和低侵袭性肾癌细胞间差异表达的cD-NA消减文库,筛选并克隆肾细胞癌转移相关基因.方法:采用涂有Matrigel胶的Transwell小室分离回收高、低侵袭性肾癌细胞,分别从高侵袭性肾癌细胞与低侵袭性肾癌细胞中提取polyA RNA,合成双链cDNA,经RsaI酶切后将高侵袭性肾癌细胞cDNA分为两组并加上接头1和接头2,再与过量低侵袭性肾癌细胞cDNA进行两次消减杂交及两次抑制性PCR,PCR产物与pMD18-T载体连接并转化JM109大肠杆菌构建成cDNA消减文库,文库扩增后随机挑取克隆进行测序及同源性分析.结果:文库共包含185个阳性克隆,随机挑取50个阳性克隆分析,其中45个克隆有插入片段.将其中15个有插入片段的克隆进行测序,表明源于7个已知基因.结论:利用抑制性消减杂交技术,从一对同源的高低转移表型差异的细胞株中获得了7条可能与肾癌转移相关的cD-NA序列,他们可能在促进肾癌转移中起到重要作用.     

转化生长因子β_1刺激肝星状细胞差异表达下调基因筛选

目的筛选并克隆转化生长因子β1(TGF-β1)刺激肝星状细胞差异表达下调基因,阐明TGF-β1导致肝纤维化的分子生物学机制。方法以TGF-β1及磷酸盐缓冲液分别刺激大鼠肝星状细胞(即实验组和对照组),提取mRNA并逆转录为cDNA,经RsaⅠ酶切后,将对照组细胞cDNA分成两组,分别与两种不同的接头衔接,再与实验组细胞cDNA进行2次消减杂交及2次抑制性PCR扩增,将产物与pGEM-Teasy载体连接,构建cDNA消减文库,并转染大肠埃希菌进行文库扩增,随机挑选克隆经PCR扩增后进行测序及同源性分析。结果成功构建了TGF-β1刺激肝星状细胞差异表达基因的cDNA消减文库。文库扩增后得到98个阳性克隆,进行菌落PCR分析,均得到200～1000bp插入片段。选取含有插入片段的35个克隆进行测序,并通过生物信息学分析获得了19种已知基因序列和2个未知功能基因。结论应用抑制性消减杂交技术成功构建了TGF-β1刺激的肝星状细胞差异表达基因的cDNA消减文库,为进一步阐明TGF-β1参与肝纤维化的分子生物学机制提供了理论依据。     

用SSH方法构建BTCC患者尿脱落细胞差异表达基因的cDNA消减文库

目的:应用抑制性消减杂交(SSH)方法构建膀胱移行细胞癌(BTCC)患者与正常人尿脱落细胞差异表达基因cDNA消减文库。方法:分别从BTCC患者与正常人尿液中提取总mRNA,用SMART技术反转录成cDNA,经过HaeⅢ酶切后将BTCC尿脱落细胞cDNA分为两组并接上接头,再与过量正常膀胱尿脱落细胞cD-NA进行两轮消减杂交及两轮抑制性聚合酶链反应(PCR),使得差异表达的DNA片段得以富集。PCR产物与T/A载体连接并转化大肠杆菌JM109构建成差异表达基因的cDNA消减文库。文库扩增后,随机挑取克隆进行酶切、测序及同源性分析。结果:PCR鉴定有317个克隆载有主要在200～900bp之间呈随机分布的插入片段,片段插入率达82.6%,证实建库成功。对20个质粒测序结果经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因。结论:该消减杂交文库质量可靠,其成功构建为进一步筛选、克隆BTCC差异表达基因提供了依据。     

青少年特发性脊柱侧弯椎间盘终板抑制消减cDNA文库的构建

用抑制消减杂交(SSH)构建组织间差异表达基因的cDNA消减文库，可进一步大批量筛选、克隆特发性脊柱侧弯表达的相关基因。     

原发性肝细胞癌消减杂交文库构建及差异表达基因筛选

目的构建人原发性肝细胞癌（HCC）消减杂交文库，筛选差异表达基因。方法以癌组织为检测者（tester）、癌旁组织为参照者（driver），应用抑制性消减杂交（SSH）方法构建消减杂交cDNA文库，随机挑选56个阳性克隆进行鉴定、测序及同源性分析。结果文库获得130个阳性克隆，鉴定96％有200～1500bp插入片断。获得的35个基因中包括已知基因26个，功能未知基因5个，新基日4个。已知基因中包含酶、细胞信号转导、基因调控因子、转录调节因子、癌基因、抗凋亡因子及细胞的黏附与生长调节因子等相关基因。结论应用SSH方法成功构建HCC差异表达基因cDNA文库，筛选出低丰度和新基因在内的HCC差异表达基因。     

膀胱癌患者尿脱落细胞抑制消减文库的构建及差异基因的初步筛选

目的 应用抑制性消减杂交方法 筛选膀胱移行细胞癌患者与正常人尿脱落细胞差异表达基因.方法 分离膀胱移行细胞癌患者与正常人尿液中总mRNA,用SMART技术反转录成cDNA,经过酶切、接头连接、两轮消减杂交及两轮抑制性PCR,使得差异表达的DNA片段得以富集.PCR产物与T/A载体连接并转化大肠杆菌XL-blue构建差异表达基因的cDNA消减文库.文库扩增后随机挑取克隆进行酶切、测序及同源性分析.结果 PCR鉴定有317个克隆载有主要在200～900bp之间呈随机分布的插入片段,片段插入率达93.2%,证实建库成功.对20个质粒测序结果 经同源性比对分析,其中20个片段源于17个已知基因,1个克隆在GenBank中未检索到与其有相似性的基因序列,表明它们可能为BTCC差异表达的新基因.结论 该消减杂交文库质量町靠,它的成功构建为进一步筛选、克隆膀胱肿瘤差异表达基因提供了依据.也为膀胱肿瘤诊断基因芯片的研究与开发奠定了基础.     

肾癌抑制性消减文库构建及差异表达基因鉴定

目的：应用抑制性消减杂交技术筛选人肾癌差异表达基因。方法：以786-0和HK-2为消减杂交对象构建人肾癌抑制性消减文库,挑选阳性克隆进行测序及Genbank BLAST分析。结果：文库包含362个有插入片段的阳性克隆,随机分析50个克隆,其中2个为新基因,另48个源于36个已知基因,这些差异表达基因与肿瘤细胞的转录、翻译、增殖、凋亡、代谢、信号转导、血管新生、膜受体表达异常等有关。结论：该文库质量可靠,筛选出包括低峰度和新基因在内的肾癌差异表达基因,为进一步研究肾癌发生、发展机制奠定了基础。     

肾癌差异表达基因GYLZ-RCC18的全长克隆及意义

目的 克隆并鉴定肾癌与下沉肾组织之间差异表达的基因。为研究肾癌发生发展机制提供新的突破口。方法 应用抑制性消减杂交技术,构建人肾癌组织与正常肾组织差异表达的ｃＤＮＡ消减文库,并从中克隆鉴定出肾癌特异表达的基因。结果：构建成功高消减效率的人肾癌组织ｃＤＮＡ消减文库,对其中１０个克隆的插入ｃＤＮＡ片段进行测序后,经基因库检索表明１０个片段均为未知新序列,其中ＧＹＬＺ－ＲＣＣ１８基因为５个拷贝,这提示以上１０个ｃＤＮＡ片段可能来自６个新基因。差异分析显示ＧＹＬＺ－ＲＣＣ１８的肾癌组织中有明显表达,而在正常肾组织中无表达,应用ＳＭＡＲＴ ＲＡＣＥ技术获得ＧＹＬＺ－ＲＣＣ１８基因的全长,并证明ＧＹＬＺ－ＲＣＣ１８是一个５′端有Ｄ３种不同剪切方式亚型的基因家族。结论 ＧＹＬＺ－ＲＣＣ１８基因是肾癌特异表达的新基因。人肾癌ｃ     

Construction of the subtractive cDNA library of injured adult and fetal rabbit skins

Objective: Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behoves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research. Methods: Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector,and then transferred into E. coli HB101. The colonies were screened afterwards. Results: The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E.coli were satisfactory. Conclusions: Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for future researches to reveal scariess healing related gene (s) and its (their) expression.     

应用抑制性消减杂交技术筛选HCV p7TP2反式调节基因

目的 应用抑制性消减杂交技术筛选HCV p7TP2反式调节基因.方法 采用实时定量PCR验证HCV p7下调p7TP2基因的表达,并应用抑制性消减杂交技术筛选并构建p7TP2下调表达基因的消减文库.用活细胞发光法检测p7TP2基因抑制Huh-7细胞系的增殖.结果 经同源性分析p7TP2下调表达的基因主要位于线粒体,并与细胞凋亡有关.p7TP2 基因转染的Huh-7细胞的活性被显著抑制.结论 p7TP2基因功能的研究为HCV致病机制的研究奠定了基础.     

Identification of novel genes expressed during metanephric induction through single-cell library screening

BACKGROUND: Development of the mature kidney is dependent on a series of inductive events between a portion of the epithelial bud at the distal end of the nephric duct and a neighboring domain of committed metanephric mesenchyme. Several genes have been identified to date that are critical in the inductive process. For example, the deletion of Bmp7 from the mouse genome results in dysgenesis or agenesis of the kidney. These findings suggest that Bmp7 controls the expression of genes important for nephrogenesis, but the identity of these genes has remained largely undetermined. METHODS: Single cells were isolated from mouse metanephric mesenchyme during the time of induction (between E11.0 and E11.5) and cDNA libraries constructed from induced and uninduced tissue. Subtractive hybridization was performed to isolate genes that were expressed during E11.5 but not E11.0. RESULTS: Using this approach, we identified eight previously known genes, three of which were known to be involved in metanephric induction, thus validating our approach, and nine novel genes. Eight of these genes were completely novel, whereas one was similar to a member of the yeast Anaphase Promoting Complex. CONCLUSIONS: Through subtractive hybridization of mouse E11.0 and E11.5 metanephric mesenchyme single-cell cDNA libraries, we have identified novel genes that are candidates for involvement in nephrogenesis through their up-regulation during the inductive process.