研究者詳細 - 洪　実

論文 - 洪　実

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A global view of gene expression in the preimplantation mouse embryo

Tanaka Tetsuya S., Ko Minoru S H

European Journal of Obstetrics Gynecology and Reproductive Biology 115 （ SUPPL. ） 2004年07月

ISSN 0301-2115

As a first step to understand preimplantation development, we performed global gene-expression profiling of morula and blastocyst using the NIA 15k mouse cDNA microarray. Gene expression levels were measured four times for blastocyst and five times for morula. Student's t-test at the 5% significance level identified 428 genes upregulated and 748 downregulated in blastocyst compared to morula. This trend was consistent with semi-quantative RT-PCR analysis of sample genes. The upregulated genes known to be involved in critical regulatory processes, included Mist1, Id2, Hd1, and Requiem; the downregulated genes included CREB-binding protein, Per3, zinc finger protein 217, Krox-25, and miwi1. Such well-characterized genes and many novel genes provide markers for early stages in development and starting materials for further functional studies.

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Gene content of the 750-kb critical region for mouse embryonic ectoderm lethal tcl-w5

Abe Kuniya, Yuzuriha Misako, Sugimoto Michihiko, Ko Minoru S H, Brathwaite Mgavi, Waeltz Paul, Nagaraja Ramaiah

Mammalian Genome 15 （ 4 ） 265 - 276 2004年04月

ISSN 0938-8990

Mice homozygous for the t^w5 allele arrest at gastrulation from defects associated with embryonic ectoderm development. The mutated gene has been genetically closely linked to the H-2K locus in the mouse MHC region, flanked by markers H-2Pb and D17Mit147. Aiming at the positional cloning of the mutated gene, we constructed a BAC contig spanning about 1 Mb of the genomic region. On the basis of our mapping and sequencing analysis of the BACs combined with public genome data, EST database searches, and gene prediction programs, we delimit the 1.06 cM of the t^w5 critical region to 750 kb, and infer 36 genes (1/20 kb) encoded in the interval. All of the 33 genes tested were confirmed as expressed in embryonic tissues by RT-PCR analyses, and in many cases by EST expression profiles as well. Thus, this highly gene-rich region is essentially totally transcribed during early development and provides priority candidates to be screened for the t^w5 embryonic lesion.

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Dynamics of global gene expression changes during mouse preimplantation development

Hamatani Toshio, Carter Mark G., Sharov Alexei A., Ko Minoru S H

Developmental Cell 6 （ 1 ） 117 - 131 2004年01月

ISSN 1534-5807

Understanding preimplantation development is important both for basic reproductive biology and for practical applications including regenerative medicine and livestock breeding. Global expression profiles revealed and characterized the distinctive patterns of maternal RNA degradation and zygotic gene activation, including two major transient waves of de novo transcription. The first wave corresponds to zygotic genome activation (ZGA); the second wave, named mid-preimplantation gene activation (MGA), precedes the dynamic morphological and functional changes from the morula to blastocyst stage. Further expression profiling of embryos treated with inhibitors of transcription, translation, and DNA replication revealed that the translation of maternal RNAs is required for the initiation of ZGA. We propose a cascade of gene activation from maternal RNA/protein sets to ZGA gene sets and thence to MGA gene sets. The large number of genes identified as involved in each phase is a first step toward analysis of the complex gene regulatory networks.

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Embryogenomics of pre-implantation mammalian development

Ko Minoru S H

Reproduction, Fertility and Development 16 （ 1-2 ） 79 - 85 2004年

ISSN 1031-3613

Pre-implantation development is marked by many critical molecular events, including the maternal to zygotic transition and the first differentiation of cells. Understanding such events is important, for both basic reproductive biology and practical applications, including regenerative medicine and livestock production. Scarcity of materials has hampered the progress of the field, but systematic genomics approaches are beginning to be applied to the study of pre-implantation development, resulting in unprecedented amounts of data about the pre-implantation process. The first step in embryogenomics is to collect and sequence cDNAs (expressed sequence tags (ESTs)) for genes that are expressed and function in these early embryos. Mouse work is the most advanced, with 140 111 ESTs derived from all stages of pre-implantation development currently available in the public sequence database. For other mammals, at present only approximately 1000 ESTs can be found in the public database, but efforts by several groups are generating cDNA libraries and ESTs. In the present review, the current status of the implementation of these investigative tools for mammalian pre-implantation embryos is discussed.

Aging of oocyte, ovary, and human reproduction

Ottolenghi Chris, Uda Manuela, Hamatani Toshio, Crisponi Laura, Garcia Jose Elias, Ko Minoru, Pilia Giuseppe, Sforza Chiarella, Schlessinger David, Forabosco Antonino

Annals of the New York Academy of Sciences 1034 117 - 131 2004年

ISSN 0077-8923

We review age-related changes in the ovary and their effect on female fertility, with particular emphasis on follicle formation, follicle dynamics, and oocyte quality. The evidence indicates that the developmental processes leading to follicle formation set the rules determining follicle quiescence and growth. This regulatory system is maintained until menopause and is directly affected in at least some models of premature ovarian failure (POF), most strikingly in the Foxl2 mouse knockout, a model of human POF with monogenic etiology (blepharophimosis/ptosis/epicanthus inversus syndrome). Several lines of evidence indicate that if the ovarian germ cell lineage maintains regenerative potential, as recently suggested in the mouse, a role in follicle dynamics for germ stem cells, if any, is likely indirect or secondary. In addition, age-related variations in oocyte quality in animal models suggest that reproductive competence is acquired progressively and might depend on parallel growth and differentiation of follicle cells and stroma. Genomewide analyses of the mouse oocyte transcriptome have begun to be used to systematically investigate the mechanisms of reproductive competence that are altered with aging. Investigative and therapeutic strategies can benefit from considering the role of continuous interactions between follicle cells and oocytes from the beginning of histogenesis to full maturation. © 2004 New York Academy of Sciences.

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The NIA cDNA project in mouse stem cells and early embryos

Carter Mark G., Piao Yulan, Dudekula Dawood B., Qian Yong, VanBuren Vincent, Sharov Alexei A., Tanaka Tetsuya S., Martin Patrick R., Bassey Uwem C., Stagg Carole A., Aiba Kazuhiro, Hamatani Toshio, Matoba Ryo, Kargul George J., Ko Minoru S H

Comptes Rendus - Biologies 326 （ 10-11 ） 931 - 940 2003年10月

ISSN 1631-0691

A catalog of mouse genes expressed in early embryos, embryonic and adult stem cells was assembled, including 250 000 ESTs, representing approximately 39 000 unique transcripts. The cDNA libraries, enriched in full-length clones, were condensed into the NIA 15 and 7.4K clone sets, freely distributed to the research community, providing a standard platform for expression studies using microarrays. They are essential tools for studying mammalian development and stem cell biology, and to provide hints about the differential nature of embryonic and adult stem cells. © 2003 Académie des sciences. Published by Elsevier SAS. All rights reserved.

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Microarray analysis of somitogenesis reveals novel targets of different WNT signaling pathways in the somitic mesoderm

Buttitta Laura, Tanaka Tetsuya S., Chen Alice E., Ko Minoru S H, Fan Chen Ming

Developmental Biology 258 （ 1 ） 91 - 104 2003年06月

ISSN 0012-1606

WNT signaling plays a major role in patterning the dermomyotome of the somitic mesoderm. However, knowledge of downstream target genes and their regulation is limited. To identify new genes involved in the development and early patterning of the somite, we performed a comparison of gene expression by microarray between the presomitic mesoderm and the 5 most recently formed somites of the mouse at embryonic day 9.5. We identified 207 genes upregulated and 120 genes downregulated in somite formation. Expression analysis and functional categorization of these genes demonstrate this to be a diverse pool that provides a valuable resource for studying somite development. Thus far, we have found three genes expressed in the dermomyotome of the early somite. Consistent with their expression patterns, these genes are transcriptional targets of WNT signals, but display differential activation by different WNTs. We further demonstrate that 1 of these genes, Troy, is a direct target of canonical WNT signaling, while the other 2 genes, Selp and Arl4, are not. Thus, our microarray study using microdissected tissues not only provides global information on gene expression during somite development, it also provides novel targets to study the inductive signaling pathways that direct somite patterning. © 2003 Elsevier Science (USA). All rights reserved.

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Plac8 and Plac9, novel placental-enriched genes identified through microarray analysis

Galaviz-Hernandez Carlos, Stagg Carole, De Ridder Gustaaf, Tanaka Tetsuya S., Ko Minoru S H, Schlessinger David, Nagaraja Ramaiah

Gene 309 （ 2 ） 81 - 89 2003年05月

ISSN 0378-1119

Microarray expression profiling of a collection of 15,000 mouse genes with placental and embryonic RNAs revealed candidates for placental-enriched genes, three of which we have confirmed and further characterized. One, Plac1, strongly expressed in all trophoblast-derived cells in the placenta, has been described earlier (Genomics 68 (2000) 305). Here we report that of the other two, Plac8 expression is restricted to the spongiotrophoblast layer during development, whereas Plac9 is weakly expressed though highly enriched in placenta. For both, cDNAs with complete open reading frames were recovered and exon-intron structures inferred from comparisons of mouse cDNA and genomic sequence. The predicted proteins (112 and 108 amino acids) both contain putative signal peptides, with a coiled-coil segment of mPLAC9 as the only other detected motif. Genomic sequence comparisons reveal that in addition to an apparent pseudogene on chromosome 1, Plac8 is expressed at mouse cytoband 5e3. It is tightly conserved in human in a syntenically equivalent ortholog at 4q21.23. Plac9 is present in a single copy on chromosome 14, with a syntenically equivalent human ortholog at 10q22.3. Putative promoter regions up to 10 kb 5′ of the transcription units for Plac1, Plac8, and Plac9 contain sites for widely-expressed transcription factors which, by analogy to other instances, may be sufficient to explain placental enrichment. © 2003 Elsevier Science B.V. All rights reserved.

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In situ-synthesized novel microarray optimized for mouse stem cell and early developmental expression profiling

Carter Mark G., Hamatani Toshio, Sharov Alexei A., Carmack Condie E., Qian Yong, Aiba Kazuhiro, Ko Naomi T., Dudekula Dawood B., Brzoska Pius M., Hwang S. Stuart, Ko Minoru S H

Genome Research 13 （ 5 ） 1011 - 1021 2003年05月

ISSN 1088-9051

Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research.

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Efficacy of 2-methoxyethoxy-modified antisense oligonucleotides for the study of mouse preimplantation development

Kimber Wendy L., Puri Nitin, Borgmeyer Carol, Ritter Donna, Sharov Alexei, Seidman Michael, Ko Minoru S H

Reproductive BioMedicine Online 6 （ 3 ） 318 - 322 2003年04月

ISSN 1472-6483

The advent of microarray technology, coupled with the availability of mouse cDNA collections derived specifically from preimplantation embryos, helps to provide global gene expression profiles for the earliest stages of development. However, to determine the functions of the large numbers of genes of interest, massive systematic functional assays such as gene 'knockdown' experiments are required. As a first step, the relative suppression of blastocyst formation by differentially-modified antisense oligonucleotides to E-cadherin was assayed. The injection of 2′-methoxyethoxy (2′-MOE)-modified oligonucleotides blocked the formation of blastocysts in two-thirds of embryos, whereas the injection of either control missense 2′-MOE-oligonucleotides, or oligonucleotides with a Morpholino modification, had no significant effect on embryonic development. Thus, the 2′-MOE-modified antisense oligonucleotides are candidates for effective examination of roles of large numbers of genes during early embryological development.

Probe design for large-scale molecular biology applications

VanBuren V., Yoshikawa T., Hamatani T., Ko M. S H

502 - 503 2003年

Large-scale molecular biology technologies such as DNA microarrays and large-scale in situ hybridization (ISH) are used to gain an appreciation of global attributes in biological tissues and cells. Although many of these efforts use cDNA probes, an approach that makes use of designed oligo probes should offer improved consistency at uniform hybridization conditions and improved specificity, as demonstrated by various oligo microarray platforms. We describe a new Web-based application that takes FASTA-formatted sequences as input, and returns both a list of the best choices for probes and a full report containing possible alternatives. Probe design for microarrays may use a scoring routine that optimizes probe intensity based upon an artificial neural network (ANN) trained to predict the average probe intensity from the physical properties of the probe and a screen for possible cross-reactivity. This new tool should provide a reliable way to construct probes that maximize signal intensity while minimizing cross-reactivity.

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Expression profiling of placentomegaly associated with nuclear transplantation of mouse ES cells

Suemizu Hiroshi, Aiba Kazuhiro, Yoshikawa Toshiyuki, Sharov Alexei A., Shimozawa Nobuhiro, Tamaoki Norikazu, Ko Minoru S H

Developmental Biology 253 （ 1 ） 36 - 53 2003年

ISSN 0012-1606

Transplantation of nuclei (NT) from engineered mouse ES cells is a potentially powerful and rapid route to create knockout mice, obviating the need for matings to obtain germ-line chimeras. However, such an application is currently impossible, because NT often results in abnormalities in embryo and placenta. Although the epigenetic instability of several imprinted genes in ES cells and ES-derived NT mice has been demonstrated, it is not clear yet what causes the abnormalities. To gain perspective on the extent and types of changes, we have done gene expression profiling for mouse placentas produced by NT of ES cells and compared them with the expression profiles of placentas produced by NT of one-cell embryos. Based on microarray studies with the NIA 15K mouse cDNA collection, we report five principal aberrant events: (1) inappropriate expression of imprinted genes; (2) altered expression of regulatory genes involved in global gene expression, such as DNA methyltransferase and histone acetyltransferase; (3) increased expression of oncogenes and growth promoting genes; (4) overexpression of genes involved in placental growth, such as Plac1; and (5) identification of many novel genes overexpressed in ES-derived NT mouse placentas, including Pitrm1, a new member of the metalloprotease family. The results indicate that placentomegaly in ES-derived NT mice is associated with large-scale dysregulation of normal gene expression patterns. The study also suggests the presence of two regulatory pathways that may lead to histologically discernable placentomegaly. The discovery of groups of genes with altered expression may provide potential targets for intervention to mimic natural regulation more faithfully in NT mice. © 2003 Elsevier Science (USA).

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Transcriptome analysis of mouse stem cells and early embryos

Sharov Alexei A., Piao Yulan, Matoba Ryo, Dudekula Dawood B., Qian Yong, VanBuren Vincent, Falco Geppino, Martin Patrick R., Stagg Carole A., Bassey Uwem C., Wang Yuxia, Carter Mark G., Hamatani Toshio, Aiba Kazuhiro, Akutsu Hidenori, Sharova Lioudmila, Tanaka Tetsuya S., Kimber Wendy L., Yoshikawa Toshiyuki, Jaradat Saied A., Pantano Serafino, Nagaraja Ramaiah, Boheler Kenneth R., Taub Dennis, Hodes Richard J., Longo Dan L., Schlessinger David, Keller Jonathan, Klotz Emily, Kelsoe Garnett, Umezawa Akihiro, Vescovi Angelo L., Rossant Janet, Kunath Tilo, Hogan Brigid L M, Curci Anna, D'Urso Michele, Kelso Janet, Hide Winston, Ko Minoru S H

PLoS Biology 1 （ 3 ） 2003年

ISSN 1544-9173

Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

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Gene expression profiling of embryo-derived stem cells reveals candidate genes associated with pluripotency and lineage specificity

Tanaka Tetsuya S., Kunath Tilo, Kimber Wendy L., Jaradat Saied A., Stagg Carole A., Usuda Masayuki, Yokota Takashi, Niwa Hitoshi, Rossant Janet, Ko Minoru S H

Genome Research 12 （ 12 ） 1921 - 1928 2002年12月

ISSN 1088-9051

Large-scale gene expression profiling was performed on embryo-derived stem cell lines to identify molecular signatures of pluripotency and lineage specificity. Analysis of pluripotent embryonic stem (ES) cells, extraembryonic-restricted trophoblast stem (TS) cells, and terminally-differentiated mouse embryo fibroblast (MEF) cells identified expression profiles unique to each cell type, as well as genes common only to ES and TS cells. Whereas most of the MEF-specific genes had been characterized previously, the majority (67%) of the ES-specific genes were novel and did not include known differentiated cell markers. Comparison with microarray data from embryonic material demonstrated that ES-specific genes were underrepresented in all stages sampled, whereas TS-specific genes included known placental markers. Investigation of four novel TS-specific genes showed trophoblast-restricted expression in cell lines and in vivo, whereas one uncharacterized ES-specific gene, Esg-1, was found to be exclusively associated with pluripotency. We suggest that pluripotency requires a set of genes not expressed in other cell types, whereas lineage-restricted stem cells, like TS cells, express genes predictive of their differentiated lineage.

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Assembly, verification, and initial annotation of the NIA mouse 7.4K cDNA clone set

VanBuren Vincent, Piao Yulan, Dudekula Dawood B., Qian Yong, Carter Carter G., Martin Patrick R., Stagg Carole A., Bassey Uwem C., Aiba Kazuhiro, Hamatani Toshio, Kargul George J., Luo Amber G., Kelso Janet, Hide Winston, Ko Minoru S H

Genome Research 12 （ 12 ） 1999 - 2003 2002年12月

ISSN 1088-9051

A set of 7407 cDNA clones (NIA mouse 7.4K) was assembled from >20 cDNA libraries constructed mainly from early mouse embryos, including several stem cell libraries. The clone set was assembled from embryonic and newborn organ libraries consisting of ∼120,000 cDNA clones, which were initially re-arrayed into a set of ∼11,000 unique cDNA clones. A set of tubes was constructed from the racks in this set to prevent contamination and potential mishandling errors in all further re-arrays. Sequences from this set (11K) were analyzed further for quality and clone identity, and high-quality clones with verified identity were re-arrayed into the final set (7.4K). The set is freely available, and a corresponding database was built to provide comprehensive annotation for those clones with known identity or homology, and has been made available through an extensive Web site that includes many link-outs to external databases and analysis servers.

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Identification, molecular characterization, and tissue expression of OVCOV1

Leach Richard E., Duniec-Dmuchowski Zofia M., Pesole Graziano, Tanaka Tetsuya S., Ko Minoru S H, Armant D. Randall, Krawetz Stephen A.

Mammalian Genome 13 （ 11 ） 619 - 624 2002年11月

ISSN 0938-8990

Key to the maternal-embryonic dialogue during early implantation are the extra embryonic trophoblast cells that play several critical roles including invasion of the maternal decidua. Cytotrophoblast proliferation and differentiation along an invasive pathway is regulated by a positive gradient of oxygen tension. We have employed transcript profiling to characterize the extra-embryonic tissues during early human implantation. We probed a cDNA library isolated from the ectoplacental cone region of stage 7.5 days post coitus mouse embryos with cDNA from hypoxic cultured human trophoblast cells. This identified the pattern of expression of a series of previously unknown genes that correlate to day 20 of human embryonic development. One EST was selected for further analysis since it is identical to the 1588-bp CGI-15 sequence assembled in silico. The human gene is organized into 10 exons 2063 bp in length. The open reading frame contains 1272 bp predicting protein composed of 365 amino acids. This gene is expressed in many tissues including those found in the reproductive tract. Its expression is regulated by oxygen tension and is unaffected by estradiol or progesterone treatment.

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EDA targets revealed by skin gene expression profiles of wild-type, Tabby and Tabby EDA-A1 transgenic mice

Cui Chang Yi, Durmowicz Meredith, Tanaka Tetsuya S., Hartung Andrew J., Tezuka Tadashi, Hashimoto Ken, Ko Minoru S H, Srivastava Anand K., Schlessinger David

Human Molecular Genetics 11 （ 15 ） 1763 - 1773 2002年07月

ISSN 0964-6906

Mutations in the EDA gene cause anhidrotic ectodermal dysplasia (EDA), with lesions in skin appendage formation. To begin to analyze EDA pathways, we have used expression profiling on 15 000-gene mouse cDNA microarrays, comparing adult mouse skin from wild-type, EDA-defective (Tabby) mice, and Tabby mice supplemented with the EDA-A1 isoform, which is sufficient to rescue multiple Tabby phenotypes. Given the sensitivity of the current microarray system, 8500 genes (60%) were estimated to be expressed, including transcription factors and growth-regulatory genes that had not previously been identified in skin; but only 24 (0.16%), one-third of them novel, showed significant differences between wild type and Tabby. An additional eight genes not included in the 15 000 gene set were shown to have expression differences by real-time RT-PCR. Sixteen of 32 affected genes were restored significantly toward wild-type levels in EDA-A1 transgenic Tabby mice. Significant up-regulation in Tabby skin was observed for several dermal matrix genes, including Col1a1, Col1a2, Col3a1 and Sparc. In contrast, down-regulation occurred for the NEMO/NF-kB pathway, already implicated in skin appendage formation, and even more markedly for a second pathway, JNK/c-jun/c-fos and their target genes, that has not previously been clearly associated with skin development. These data are consistent with the regulation of the NF-kB pathway by EDA, and support its involvement in the regulation of the JNK pathway as well.

Evaluation and comparison of clustering algorithms in analyzing ES cell gene expression data

Chen Gengxin, Jaradat Saied A., Banerjee Nila, Tanaka Tetsuya S., Ko Minoru S H, Zhang Michael Q.

Statistica Sinica 12 （ 1 ） 241 - 262 2002年01月

ISSN 1017-0405

Many clustering algorithms have been used to analyze microarray gene expression data. Given embryonic stem cell gene expression data, we applied several indices to evaluate the performance of clustering algorithms, including hierarchical clustering, k-means, PAM and SOM. The indices were homogeneity and separation scores, silhouette width, redundant score (based on redundant genes), and WADP (testing the robustness of clustering results after small perturbation). The results showed that the ES cell dataset posed a challenge for cluster analysis in that the clusters generated by different methods were only partially consistent. Using this data set, we were able to evaluate the advantages and weaknesses of algorithms with respect to both internal and external quality measures. This study may provide a guideline on how to select suitable clustering algorithms and it may help raise issues in the extraction of meaningful biological information from microarray expression data.

Embryogenomics

Ko Minoru S H

Trends in Biotechnology 19 （ 12 ） 511 - 518 2001年12月

ISSN 0167-7799

Fundamental questions in developmental biology are: what genes are expressed, where and when they are expressed, what is the level of expression and how are these programs changed by the functional and structural alteration of genes? These questions have been addressed by studying one gene at a time, but a new research field that handles many genes in parallel is emerging. The methodology is at the interface of large-scale genomics approaches and developmental biology. Genomics needs developmental biology because one of the goals of genomics - collection and analysis of all genes in an organism - cannot be completed without working on embryonic tissues in which many genes are uniquely expressed. However, developmental biology needs genomics - the high-throughput approaches of genomics generate information about genes and pathways that can give an integrated view of complex processes. This article discusses these new approaches and their applications to mammalian developmental biology.

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Assignment of OVCOV1 (alias CGI-15) to human chromosome 20 band q13.1→q13.2 by fluorescent in situ hybridization

Leach Richard, Duniec-Dmuchowski Z., Tanaka T., Ko M. S., Krawetz S. A.

Cytogenetics and Cell Genetics 94 （ 3-4 ） 252 - 253 2001年

ISSN 0301-0171

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