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 clones 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.

应用抑制性消减杂交（suppression subtracted hvh ridization, SSH）技术，分别构建2个不同淋巴道转移能力小鼠肝癌细胞株的SSH文库．高转移文库以高转移能力细胞株Hca-F为检测子（tester），同源的低转移能力细胞株Hca-P为驱赶子（driver）：低转移文库则相反．随机筛选2个文库阳性克隆进行测序。并在GenBank数据库中进行l司源性比较。成动构建高、低淋巴道转移能力小鼠肝癌细胞株SSH文库，高、低转移文库中分别包含995个及967个阳性克隆：其中．95%的阳性克隆含有300bp-1OOObp不等的插入片段：随机测序显示，文库中含有已知的基凶。ESTs及与任何序列无例源性的新基凶片段。

AIM: To identify genes differentially expressed in mouse hepatocarcinoma ascites cell line with low potential of lymphogenous metastasis. METHODS: A subtracted cDNA library of mouse hepatocarcinoma cell line with low potential of lymphogenous metastasis Hca-P and its synogenetic cell line Hca-F with high metastatic potential was constructed by suppression subtracted hybridization (SSH) method.The screened clones of the subtracted library were sequenced and GenBank homology search was performed. RESULTS:Fifteen differentially expressed cDNA fragments of Hca-P were obtained which revealed 8 known genes,4 expressed sequence tags (ESTs) and 3 cDNAs showed no homology. CONCLUSION: Tumor metastasis is an incident involving multiple genes.SSH is a useful technique to detect differentially expressed genes and an effective method to clone novel genes.

AIM: In order to obtain lymphogenous metastasisassociated genes,we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.METHODS:Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA.In vitro transcription double-stranded cDNA was labeled with biotin (i.e.biotin-labeled cRNA,used as the probe).The cRNA probes hybridized with Affymetrix GeneChip MOE430A (containing 22 690 transcripts,including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner.The results were then analyzed by bioinformatics. RESULTS: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold.Among the total 4 371 ESTs,17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis,the 110 genes were further classified into two groups:differential biological process profile and molecular function profile. CONCLUSION: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion,signal transduction, cell motility, transport, microtubule-based process,cytoskeleton organization and biogenesis, cell cycle, transcription, chaperone activity, motor activity, protein kinase activity, receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation. Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments. ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.