Construction of a metastasis-associated gene subtracted cDNA library of human colorectal carcinoma by suppression subtraction hybridization

AIM:To construct a differentially-expressed gene subtractedcDNA library from two colorectal carcinoma (CRC) cell lineswith different metastatic phenotypes by suppressionsubtractive hybridization.METHODS:Two cell lines of human CRC from the samepatient were used.SW620 cell line showing highlymetastatic potential was regarded as tester in the forwardsubtractive hybridization,while SW480 cell line with lowlymetastatic potential was treated as tester in the reversehybridization.Suppression subtractive hybridization (SSH)was employed to obtain cDNA fragments of differentiallyexpressed genes for the metastasis of CRC.These fragmentswere ligated with T vectors,screened through the blue-white screening system to establish cDNA library.RESULTS:After the blue-white screening,235 white cloneswere picked out from the positive-going hybridization and232 from the reverse.PCR results showed that 200-700 bpinserts were seen in 98% and 91% clones from the forwardand reverse hybridizations,respectively.CONCLUSIONS:A subtractive cDNA library of differentiallyexpressed genes specific for metastasis of CRC can beconstructed with SSH and T/A cloning techniques.     

人源性大肠癌cDNA噬菌体表达文库的构建及鉴定

目的　构建人源性大肠癌cDNA噬菌体表达文库。方法　从人大肠癌组织中提取总RNA ,RT PCR反转录合成cDNA第 1链 ,用长距离PCR技术合成cDNA第 2链 ,除去小于 5 0 0bp的小片段后与λTriplEx2噬菌体连接 ,体外包装 ,构建成cDNA噬菌体表达文库。转染E .coliXL1 Blue大肠杆菌 ,滴定文库的滴度 ,确定文库容量大小 ,测定重组率。用EXTagTM酶做PCR和SfiⅠ酶切鉴定插入的cDNA片段大小。结果　构建成含 2 .39× 10 6pfu/ml重组子的人大肠癌cDNA噬菌体表达文库 ,重组率为 97.5 %。插入的外源cDNA片段大小为 5 0 0bp～ 4kb ,平均长约 1.4kb。 结论　成功构建高质量人源性大肠癌cDNA噬菌体表达文库 ,适合于免疫筛选cDNA克隆的人大肠癌相关抗原基因。     

正常肝组织与肝癌组织差异表达基因消减cDNA文库的构建

目的 构建人正常肝组织与肝癌组织差异表达基因的消减cDNA文库。方法 采用新近建立的抑制消减杂交技术,以癌旁正常肝组织及肝癌组织作为对比材料,分离肝癌组织中不表达或低表达基因的cDNA片段,将其与T载体进行T/A连接构建文库,将连接产物用电穿孔法转化大肠杆菌进行文库扩增后,随机挑取100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆进行酶切鉴定。结果 扩增消减cDNA文库获得4000余个白色阳性克隆,随机挑取的100个白色克隆经酶切后均有200-600bp的插入片段。结论 用SSH法及T/A克隆技术成功构建了正常肝组织与肝癌组织差异表达基因的消减cDNA文库,该文库的建立为进一步筛选、克隆肝癌组织中失活或低表达的新的抑癌基因奠定了基因。     

胰腺癌抑制消减cDNA文库的构建及质量分析

目的构建人胰腺癌抑制消减cDNA文库.方法从来源于同一标本的胰腺癌和癌旁正常组织分离poly(A)+ RNA,经反转录后,利用抑制消减杂交(SSH)方法,通过两轮杂交和两次抑制PCR构建了两种组织间差异表达基因的cDNA消减文库.结果挑取500个克隆进行PCR扩增检测是否有插入片段,结果显示其中 457个克隆有插入片段,片段大小范围为 250～750 pb.结论应用消减杂交的方法,再经适当的改进,在去除相同遗传背景的条件下,可以建立较特异的胰腺癌cDNA消减文库,该文库为进一步批量筛选胰腺癌发生的相关基因群并克隆胰腺癌相关表达基因,研究其胰腺癌发生的分子机理与生物学特性间的关系奠定了基础.     

胰腺癌抑制消减cDNA文库的构建及质量分析

目的：构建人胰腺癌抑制消减cDNA库。方法：从来源于同一标本的胰腺癌的和癌旁正常组织分离poly(A)^ RNA,经反转录后，利用抑制消减杂交（SSH）方法，通过两轮杂交和两次抑制PCR构建了两种组织间差异表达基因的cDNA消减库。结果：挑取500个克隆进行PCR扩增检测是否有插入片段，结果显示其中457个克隆有插入片段，片段大小范围为250－750pb。结论：应用消减杂交的方法，再经适当的改进，在去除相同遗传背景的条件下，可以建立较特异的胰腺癌cDNA消减库，该库为进一步批量筛选胰腺癌发生的相关基因群并克隆胰腺癌相关表达基因，研究其胰腺癌发生的分子机理与生物学特性间的关系奠定了基础。     

人胃癌组织定向cDNA文库的构建

目的快速构建成人胃癌组织cDNA文库。方法从人胃癌组织分离总RNA,以含有Sfi IB酶切位点的oligo(dT)引物合成第1链cDNA,以含Sfi IA酶切位点的SMART寡核苷酸为引物经LD-PCR扩增双链cDNA,双链cDNA经Sfi I(IA & IB)酶切,以CHROMA SPIN+TE-1000柱分级分离cDNA,收集符合需要的cDNA片段并纯化,随后将之与λTripIEx2载体连接经体外包装成噬菌体cDNA文库。结果经检测共获得2.73×10~6个重组子,重组率约为94%,插入片段大小平均为1.2 kb。结论用SMART方法构建的胃癌组织cDNA文库质量较高,为以后筛选胃癌相关基因奠定了基础。     

人胰岛细胞瘤cDNA文库的构建

采用一种简单高效的互补脱氧核糖核酸（ｃＤＮＡ）克隆技术制备人胰岛细胞瘤的ｃＤＮＡ文库。方法的主要过程如下：以人胰岛细胞瘤的Ｐｏｌｙ（Ａ）＾＋－ＲＮＡ为模板，以含ＮｏｔＩ切点的ｏｌｉｇｏ－（ｄＴ）１５为引物，在反转录酶作用下合成第一股单链ｃＤＮＡ；用Ｅ．ｃｏｌｉ ＲＮａｓｅＨ除去Ｐｏｌｙ（Ａ）＾＋－ＲＮＡ，并以Ｅ．ｃｏｌｉ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ ＩＥ．ｃｏｌｉ ＤＮＡ Ｌｉｇａｓｅ和Ｔ４ＤＮ     

抑制消减杂交法筛选无症状乙型肝炎病毒携带者差异表达基因

乙型肝炎病毒（HBV）携带者中,大多数是婴幼儿期感染,往往终生携带HBV,但正常成人初次感染后大多能清除病毒.除病毒因素外,主要是机体的免疫因素决定感染结局[1].机体对HBV的免疫应答受许多基因的调节,我们推测正是由于在正常人和HBV携带者之间存在的免疫相关基因表达的差异才导致了不同的感染结局.本研究旨在探讨HBV携带者与正常人外周血单个核细胞（PBMC）差异表达的基因,有助于阐明HBV携带发生发展机制及设计新的治疗靶标.     

人原发性肝癌组织cDNA文库的构建及鉴定

目的构建人原发性肝癌组织cDNA文库,筛查与原发性肝癌发生特异相关的基因,为探讨原发性肝癌的发病机制及基因治疗奠定基础。方法提取人原发性肝癌组织总RNA并纯化其mRNA;逆转录合成单链cDNA,长距离聚合酶链反应(PCR)合成双链cDNA;PCR产物经蛋白酶K水解并纯化后,进行SfiⅠ酶酶切;将酶切产物进行分级分离,回收0.4 kb以上的组分,并与λTriplEx2载体连接;将连接产物经体外蛋白包装,产生未扩增文库;检测未扩增文库的滴度和重组效率后,进行文库扩增;检测扩增后文库的滴度和重组效率,随机挑取14个噬菌斑,用载体克隆位点两端的通用引物进行PCR扩增,以检测所构建的cDNA文库的质量。结果未扩增文库的滴度为1.37×106pfu/ml,重组效率为97.46%;扩增后文库滴度为1.28×109pfu/ml,重组效率为99.06%;插入片段平均长度为0.95 kb,长度在1.0~2.0 kb的4个,0.75~1.0 kb的4个,0.5~0.75 kb的6个。结论已成功构建一个乙型肝炎肝硬化基础上发生的人原发性肝癌组织cDNA文库。     

Differential proteomic analysis of human colorectal carcinoma cell lines metastasis-associated proteins

Purpose Metastasis is a common phenomenon and the major lethal cause of colorectal carcinoma (CRC). To better comprehend the mechanism underlying CRC metastasis and to search for potential markers for predicting CRC metastasis, two CRC cell lines with different metastatic potentials, SW480 and SW620, were investigated by phenotypic analyses and proteomics technologies. Methods The surgical orthotopic implantation (SOI) technique was originally used to develop a reproducible colorectal cancer model in nude mice with stable tumor growth and metastasizing course. Furthermore, differential proteome analysis of two CRC cell lines was conducted using two-dimensional (2-D) gel electrophoresis combined with matrix-assisted laser desorption/time of flight (MALDI-TOF) mass spectrometry. Results Among the differential spots, 12 protein spots (11 proteins) were further identified using in-gel tryptic digestion and peptide mass fingerprinting (PMF). Interestingly, most of these proteins we identified have been reported to be associated with distinct aspect of tumor metastasis to some extent. Our immunohistochemistry assays of colorectal cancer revealed that heat shock protein (HSP) 27 overexpression relates to metastatic behavior of CRC cell. Conclusions The protein profile between two colorectal cell lines with different potential metastasis displayed obvious differences. The results imply that various different proteins may lead to CRC metastasis together. HSP27 overexpression played an important role in metastasis and progression of CRC.     

马尔尼菲青霉酵母相消减cDNA文库的构建及初步筛选

目的构建马尔尼菲青零酵母相抑制性消减cDNA文库，寻找其在酵母相中的差异表达基因。方法分别提取马尔尼菲青零菌丝相和酵母相的总RNA并合成cDNA，然后应用抑制性消减杂交技术（SSH）。以酵母相为tester（检测子），菌丝相为driver（驱赶子），连接不同的接头，通过两轮杂交和两次抑制性PCR后，将产物与T载体连接并转染大肠杆菌。经PCR鉴定，共得到480条插入片段。序列分析和同源性比较表明一些基因与细胞壁抗原、转运蛋白、氧化还原酶等具有同源性。结果成功构建了一个以酵母相为tester（检测子）的抑制性消减cDNA文库。结论所构建的cDNA消减文库为进一步筛选马尔尼菲膏零致病相关基因奠定了基础。     

Construction and selection of subtracted cDNA library of mouse hepatocarcinoma cell lines with different lymphatic metastasis potential

AIM: In order to elucidate the molecular mechanism of lymphatic metastasis of hepatocarcinoma, we detected the difference of gene expression between mouse hepatocarcinoma cell lines Hca-F and Hca-P with different lymphatic metastasis potential.METHODS: cDNA of Hca-F cells was used as a tester and cDNA of Hca-P cells was used as a driver, cDNAs highly expressed in Hca-F cells were isolated by the suppression subtractive hybridization (SSH) method. The isolated cDNA was cloned into T/A cloning vector. The ligation products were transformed into DH5 α competent cells. Individual dories were randomly selected and used for PCR amplification.Vector DNA from positive clones was isolated for sequencing.RESULTS: There were 800 positive clones in amplified subtracted cDNA library. Random analysis of 160 clones with PCR showed that 95% of the clones contained 100-700 bp inserts. Analysis of 20 sequenced cDNA clones randomly picked from the SSH library revealed 4 known genes(mouse heat shock protein 84 ku, DNA helicase, ribosomal protein S13 ,ethanol induced 6 gene) and 3 expressed sequence tags (ESTs). Four cDNAs showed no homology and presumably represent novel genes.CONCLUSION: A subtracted cDNA library of differentially expressed genes in mouse heptocarcinoma cell lines with different lymphatic metastasis potential was successfully constructed with SSH and T/A cloning techniques. The library is efficient and lays a solid foundation for searching new lymphatic metastasis related genes. The expression of mouse heat shock protein gene, DNA helicase and other 4 novel gene may be different between mouse heptocarcinoma cell lines with different lymphatic metastasis potential.     

人胰腺癌抑制消减cDNA文库的构建

目的应用抑制消减杂交技术(SSH)构建胰腺癌和正常胰腺组织间差异表达的抑制消减cDNA文库。方法分别提取胰腺癌(tester)和癌旁正常胰腺组织(driver)中的总RNA和mRNA．合成双链cDNA，经RsaI酶切后，将胰腺癌双链cDNA分为两组，分别加上不同的接头，再与正常胰腺组织cDNA进行两次消减杂交及两次抑制性PCR，分离出胰腺癌差异表达基因的cDNA片段。将该差异表达片段克隆至T／A载体，并转化大肠杆菌TOP10F’，经蓝白斑筛选后，再用PcR方法筛选阳性克隆，从而构建胰腺癌抑制消减cDNA文库。结果文库扩增后得到257个白色克隆，随机挑取50个阳性克隆进行PCR扩增分析，其中47个克隆有插入片段．克隆阳性率为94％，片段大小主要集中在300～600bp之间。结论成功构建了人胰腺癌抑制消减cDNA文库，为进一步筛选、克隆胰腺癌特异性表达基因奠定了基础。