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。     

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.     

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。     

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。     

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     

抑制性消减杂交技术克隆肾癌差异表达基因

目的 应用抑制性消减杂交技术构建人吕组织与正常肾组织差异表达的ｃＤＮＡ消减文库，并从中克隆鉴定出蛑癌特异性表达的新基因。方法 分别从肾癌及正常肾组织中提取ｍＲＮＡ并合成ｃＤＮＡ，经酶切后将肾癌ｃＤＮＡ分为两组，分别与两种不贩接头衔接，再与正常肾组织（ｃＤＡＮ）进行两次的消减杂交及两次抑制性ＰＣＲ产物与Ｔ／Ａ载体连接构建成功ｃＤＮＡ消减文库，并转染大肠杆菌进行文库扩增，随机挑取克隆进行酶切、测序分析     

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.     

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。     

Suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines

Small Cell Lung Cancer (SCLC), a clinically aggressive cancer, accounts for approximately 25% of primary lung cancers. We carried out suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, between the human classic, NCI-H69 and variant, more aggressive NCI-N417 SCLC cell lines to isolate and characterize variable expression of genes, which may be responsible for differential degree of tumorigenicity of SCLC. Using NCI-N417 as a tester, we obtained 28 differentially expressed cDNA clones from a total of 60 arbitrarily picked clones. Among the 28 cDNA clones, 4 were unknown genes, 2 were fatty acid binding protein (FABP) with specific identification of mRNA for mammary-derived growth inhibitor (MDGI), 1 was human alpha-enolase, 4 were ribosomal proteins, 2 were structural genes, vimentin and moesin (membrane-organizing extension spike protein), and 9 were homologous with murine leukemia viruses, whereas 2 others had enhanced expression in NCI-H69 and A549 cell lines, and 4 were cell surface proteins and murine type C retrovirus. Expression of FABP/MDGI was significantly high in NCI-H417, which may influence mitosis and cell growth as implicated in other tissues, contrary to the conclusion drawn for the role of MDGI in human breast cancer. Higher expression of ribosomal proteins in NCI-N417 compared to NCI-H69 may have a role in differential tumorigenicity and metastatic ability. Further, we obtained 14 differentially expressed cDNA clones by reversing the tester and driver, using NCI-H69 as a tester. Of these 14 differential cDNAs, 5 were unknown genes, 2 were specific for keratins, others had similarities with protease inhibitor, human BAC clone, Alu RNA binding protein, and tumor expression-enhanced gene. Characterization of these differentially expressed cDNA clones will provide useful information in understanding of the genes responsible for differential tumorigenicity of SCLC.     

Cloning of differentially expressed genes following lipopolysaccharide stimulation in human umbilical vein endothelial cells

Objective: To clone the differentially expressed genes in human umbilical vein endothelial cells (HUVEC)stimulated by lipopolysaccharide (LPS). Methods : Two-directional ( forward and backward)suppression subtractive hybridization ( SSH ) was performed on HUVEC cultured in either standard media or treated for 6 hours with LPS (100 ng/ml). To restrict the number of false-positive clones, colony dot hybridization was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced. Results: These analyses have identified both novel and known genes whose expression is influenced by LPS.The known genes include a group related to proinflammatory events, a group related to cellular apoptosis and proliferation, a group related to protein synthesis and cytoskeletal rearrangment, and a group related to energy metabolism and signal transduction. Conclusions: SSH is a powerful technique of high sensitivity for the detection of differential gene expression in HUVEC stimulated by LPS.     

Establishment and characterization of seven human renal cell carcinoma cell lines

OBJECTIVE: To establish human renal cell carcinoma (RCC) cell lines, and to investigate the cell phenotypes and molecular characteristics of human RCC cell lines and their corresponding tumour tissues. MATERIALS AND METHODS: Seven human RCC cell lines from pathologically proven RCCs were established. The histopathology of the primary tumours, in vitro growth characteristics and status of tumour suppressor genes, mismatch repair genes and microsatellite instability (MSI) were examined in cell lines and their corresponding tumour tissues. Five of the cell lines were derived from clear cells (SNU-228, -267, -328, -349, and -1272), one from granular cells (SNU-482), and one from mixed clear and granular cell types (SNU-333). The mutational status was compared for von Hippel-Lindau (VHL), p53, TGF-beta type II receptor (TGF-betaRII), hMSH2, and hMLH1 genes in the cell lines and their corresponding tumour tissues. The MSI status of the cell lines was determined by screening for adenine repeat sequences, e.g. BAT-25, BAT-26, and BAT-40. RESULTS: All lines showed different doubling times and were confirmed by DNA fingerprinting analysis to be unique. Contamination by mycoplasma or bacteria was excluded. In two cell lines (SNU-349 and -1272) and their tumour tissues, mutations in the VHL gene were found. The SNU-267 line had a frameshift mutation in the p53 gene. A missense mutation of the TGF-betaRII gene was detected in the SNU-1272 line and the corresponding tissue. Analysis of the repeat sequences showed one cell line (SNU-349) to have MSI and the other six to have microsatellite stability. As MSI is a hallmark of the inactivation of mismatch repair genes, the presence of hMSH2 and hMLH1 mutations was investigated in all seven cell lines. An inactivating homozygous single base-pair deletion of the hMLH1 gene was found only in the SNU-349 cell line and corresponding tissue. Moreover, a frameshift mutation within an 8-bp polyadenine repeat present in the hMSH3 coding region was found only in the MSI cell line and tumour tissue. CONCLUSION: These newly established RCC cell lines should provide a useful in vitro model for studies related to human RCC. The SNU-349 cell line should be especially useful for studies of MSI and mismatch repair-defective RCCs.     

人精子有效抗原基因表达克隆的研究

我们从人睾丸组织中提取ＲＮＡ，并进一步纯化出ｍＲＮＡ；以此为模板，在反转录酶和ＤＮＡ多聚酶的作用下，合成ｃＤＮＡ；将ｃＤＮＡ与λｇＩＩ载体重组后转染大肠杆菌，构建人睾丸ｃＤＮＡ表达文库。运用遗传显色法和噬菌斑原位杂交法鉴定表达文库后，用兔抗人精子抗体筛选人精子抗原基因表达克隆（ＨＳＧ）。对ＨＳＧ表达抗原（ＨＳＧＡｇ）和特异抗体（ＨＳＧＡｂ）进行纯化和抗生育效应的测定。结果显示：（１）人睾丸ｃＤＮＡ表达文库容量为１．８２Ｘｌ０６ｐｆｕ，遗传显色法示重组率为６７％，噬菌斑原位杂交法示重组率为５１％。（２）经兔抗人精子抗体筛选２Ｘｌ０４ｐｆｕ，得８株人精子抗原基因表达克隆。（３）人血清、兔血清ＨＳＧ２Ａｂ和兔血清ＨＳＧ８Ａｂ对人精子具有补体依赖细胞毒作用。（４）兔血清ＨＳＧ３Ａｂ能阻断人精子在顶体反应时顶体后区ＣｏｎＡ受体的暴露。结果提示，ＨＳＧ２、ＨＳＧ３和ＨＳＧ８克隆的表达抗原是精子有效抗原，能作为精子免疫避孕疫苗的候选成分。