用基因芯片结合荧光差异显示—PCR筛选和识别胃腺癌转移相关的基因

使用来源于同一个胃腺癌病人的原发灶RF－1（ATCC编号：CRL－1864）和转移灶RF－48细胞系（ATCC编号,CRL－1863）作为研究肿瘤转移分子机制的模型,RF－1（实验组）和RF－48（对照组）的mRNA通过逆转录方法,将Cy3和Cy5两种荧光染料分别标记到两种细胞的cDNA上,制备成cDNA探针,并与表达谱芯片（双点4096条基因）进行杂交与扫描,重复2次实验,利用计算机数据处理判断基因是否在上述两种细胞中有表达差异,共筛选出差异表达的基因共138条,其中81条在RF－48细胞中表达明显上调,57条在RF－48细胞中表达显著下调,同时也通过荧光差异显示－PCR（FDD－PCR）技术,克服了45个涉及胃腺癌转移相关基因,包括未被发现的基因3个,在两种筛选方法中都存在差异表达的基因共有7条,对部分可能与肿瘤志移机制有关的差异表达基因的作用进行了分析和讨论,基因芯片技术可高通量,大规模地研究基因表达水平,FDD－PCR技术可克隆出未发现的新基因,二者结合,初步筛选出与转移相关的基因,有助于揭示胃腺癌转移的分子机制。     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

Use of specific single stranded DNA probes cloned in M13 to study the RNA synthesis of four temperature-sensitive mutants of HK/68 influenza virus.

Specific single stranded DNA probes have been obtained for both influenza virion RNA (vRNA) and complementary RNA (cRNA) by cloning a hemagglutinin gene fragment in the single stranded DNA phase M13. These probes were used for hybridization with the total labeled RNA from cytoplasmic extracts of infected cells. MDCK cells were infected with temperature-sensitive mutants of influenza HK/68 and the production of the virus specific RNA species was analysed at both permissive and restrictive temperatures. Results show that two NP mutants which undergo intracistronic complementation exhibit two different phenotypes at the non permissive temperature: ts2C is poly A cRNA and vRNA negative whereas ts463 is RNA positive. Two mutants of P genes were also analysed and we discuss the relationship existing between the synthesis of the three RNA species especially between poly A and non poly A cRNA.     

不同生存条件下东方田鼠肝脏基因表达谱分析

目的比较实验室条件下饲养的东方田鼠和野外捕捉东方田鼠肝脏的基因表达差异,寻找可能参与肝脏病变的关键基因。方法以实验室条件饲养的东方田鼠和野外捕捉东方田鼠为研究对象,分别抽提RNA,逆转录成cDNA,体外转录为cRNA并进行片段化;利用表达谱芯片分别进行杂交,扫描后筛选差异基因,并应用real-time PCR方法对部分基因的表达水平进行进一步测定,验证芯片数据的结果。结果实验室饲养东方田鼠肝组织与野外捕捉东方田鼠相比,共有99个基因和41个EST差异表达。其中参与机体代谢的基因占主导,约占35.4%;其次为参与信号通路的基因,约占24.2%;参与细胞周期和免疫的基因分别占6.1%和3.0%。结论利用基因表达谱芯片初步筛选了可能参与东方田鼠脂肪肝形成过程的基因,发现机体代谢通路的基因占主导,肝脏中细胞色素家族基因表达差异明显。     

Characterization of a novel glycoprotein isolated from the basement membrane matrix of the Engelbreth-Holm-Swarm tumor

A previously undescribed protein has been isolated and purified from the extracellular matrix of the Engelbreth-Holm-Swarm (EHS) tumor, a murine tumor that synthesizes an extensive matrix composed of basement membrane molecules. Molecular characterization of the molecule determined that it is a glycoprotein with internal disulfide bonds and an isoelectric point of 6.0. Electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the glycoprotein migrated as a diffuse band with a molecular weight of approximately 72,000-80,000. The amino acid composition was significantly different from known basement membrane components. Polyclonal antibodies that specifically recognize the glycoprotein localized it to the kidney glomerular basement membrane. These antibodies did not cross-react with either known basement membrane components (laminin, type IV collagen, and heparan sulfate proteoglycan), with 70K "culture shock" protein or with components of normal mouse serum (including mouse transferrin, albumin, or alpha-fetoprotein), when analyzed by "Western" immunoblots. Our data indicate that the glycoprotein is synthesized by the EHS tumor cells and is present at relatively high levels in the EHS tumor matrix.     

基因文库的鉴定和应用

由于草菇是一种同宗结合的食用真菌,这给草菇的杂交育种带来了一定的困难。本文在建立草菇部分基因文库的基础之上,对草菇的基因文库进行了鉴定。在草菇基因文库中任意抽取72个克隆,利用专一的PCR方法,测出在草菇基因文库中,草菇基因组DNA的平均大小为1156个碱基对。在基因文库中任意选择53个克隆,利用专一的PCR进行DNA扩增以及dig非同位素标记,用于和草菇基因组DNA杂交。在测试的53个克隆中有8％的高度重复序列,36％中度重复序列和56％的低度重复序列。     

Purification and tissue distribution of a small protein (BM-40) extracted from a basement membrane tumor

A novel acidic glycoprotein, BM-40, with Mr = 40,000, was purified from the basement-membrane-producing mouse EHS tumor and characterized with regard to its unique chemical and antigenic properties. It was obtained from the tumor in a neutral salt-soluble form or as a component requiring extraction with 6M guanidine X HCl. This protein could also be identified in many other tissue extracts and cell and tissue cultures. The most intact form of BM-40 consists of a single polypeptide chain which undergoes limited proteolysis during extraction and purification. BM-40 exists in most tissues in stoichiometric amounts compared to other basement membrane proteins (laminin, nidogen) and is secreted by various teratocarcinoma and epithelial cells. It can be visualized by immunofluorescence in the extracellular matrix of the EHS tumor and Reichert's membrane. Other tissues which contain extractable BM-40 were negative in immunofluorescence.     

Detection of oligodendrocytes in tissue sections using PCR synthesis of digoxigenin-labeled probes.

Oligodendrocytes, the myelin-forming cells in the central nervous system, were visualized with excellent resolution at the light microscopic level using in situ hybridization (ISH). Digoxigenin (Dig)-tagged probes were synthesized and efficiently labeled by PCR. Specific probes to myelin genes were made by RT from brain total RNAs, followed by PCR with designed specific primers in the presence of Dig-11-dUTP. Probes specific to proteolipid protein (PLP), PLP and its isoform DM20 (PLP/DM20), and myelin oligodendrocyte glycoprotein (MOG) were synthesized and labeled. ISH was then applied on vibratomed tissue sections from mouse brains. Despite a low expression of MOG-specific and PLP-specific mRNAs in adult and newborn mouse brains, an oligodendrocyte population was detected. The specificity of Dig-labeled probes was confirmed with the double labeling of carbonic anhydrase II (CA II) and glial fibrillary acidic protein (GFAP) immunocytochemistry and ISH. This versatile and easy method for synthesis and labeling of specific probes to oligodendrocytes can be also applied to detect many other mRNAs in the nervous system and in other tissues.     

Extracellular matrix and mouse mammary cell function: Comparison of substrata in culture

Summary Cultured mammary cells depend on interaction with a substratum for functional differentiation, even in the presence of lactogenic hormones. Protein synthesis and secretion by mouse mammary epithelial cells on floating collagen gels and (EHS) matrix were compared. Cells were prepared by collagenase digestion of tissue from mid-pregnant mice. Protein synthesis was consistently greater in cells attached to EHS matrix, and was associated with proportionately higher rates of protein secretion into culture medium. Cells on EHS secreted protein into a luminal space formed within multicellular alveoluslike structures. Luminal secreted protein, extracted by EGTA treatment of cells in situ, constituted up to 40% of total secreted radiolabeled protein for cells on EHS matrix. The EGTA extract contained a higher proportion of casein and lactoferrin, whereas transferrin was predominately in the medium. This indicated that cells on EHS matrix had become polarized and were secreting proteins vectorially. In contrast, EGTA treatment of cells on floating collagen gels released virtually no radiolabeled protein, showing that mammosphere formation was a property of cells on EHS. These biochemical observations were supported by ultrastructural evidence. In EHS cultures, the proportion of secreted protein in the luminal fraction, but not the distribution of secreted proteins, changed with time. This suggests that there may be leakage out of the lumen, or intraluminal degradation of protein after secretion. Nevertheless, the results suggest that cellular organization into mammospheres on EHS matrix promotes synthetic and secretory activity. This system provides a useful model for investigation of the regulation of milk secretion.     

Cell-free translation products of basement membrane RNA from the EHS tumor

The biosynthetic products of the Engelbreth-Holm-Swarm (EHS) tumor and the cell-free translation products of EHS tumor cell RNA were characterized. Six distinct gene products (three laminin polypeptides, entactin/nidogen, and two collagen IV chains) comprising the basement membrane matrix were identified by a combination of proteolytic digestion and immunologic techniques. Analysis of the cell-free translation products using EHS tumor RNA precipitated by anti-laminin serum confirms earlier evidence that there are at least two B chains encoded by different genes. The anti-laminin serum also recognized entactin/nidogen, which was further identified by specific immunoprecipitation with anti-entactin serum. Radiolabeled laminin A chains, synthesized by the EHS tumor in organ culture, were also identified by the anti-laminin serum but were not detected among the cell-free translation products of EHS tumor RNA. Pulse-chase studies of EHS tumor in organ culture as well as in vitro translation of EHS tumor RNA suggest that the precursor forms of alpha 1(IV) and alpha 2(IV) collagen chains are nearly identical in size, with apparent molecular weights of 170,000. The mRNAs encoding these two polypeptides migrate differently on sucrose gradients. It is likely that glycosylation and hydroxylation of collagen IV account for the major differences in molecular weight of mature alpha 1(IV) and alpha 2(IV) chains in the EHS tumor matrix.