人胆管癌细胞分化相关基因cDNA克隆与功能研究

Cholangiocarcinoma is a rare but highly malignant primary neoplasm of biliary tract, which has a high incidence in China and Far East. Early diagnosis is difficult and current therapies are ineffective for the advanced disease. Although the molecular pathogenesis of cholangiocarcinoma is still poorly understood, the clinicopathological evidence presented supports a strong positive correlation between cancer differentiation levels and prognosis in human cholangiocarcinoma. Cholangiocarcinoma differentiation-related gene may play an important role in the cholangiocarcinogenesis and development of cholangiocarcinoma, and may be a useful biomarker for the early-stage diagnosis and prognostication of prognosis and a potential target of genetic therapeutic strategy. This study was designed to clone the cDNA sequence of the novel cholangiocarcinoma differentiation-related gene. A novel sample database containing about 60 cholangiocarcinoma patients' fresh tissues was made in the PLA hospital. A cDNA microarray with 14802 genes was used to describe the differential gene expressional profile of differential phenotype human cholangiocarcinoma cells for the first time. About 754 genes were observed remarkably differentially expressed，including 455 known gene, such as BTGl gene, MUC18 gene, GRB2 gene, angiogenin gene, KGF gene and so on. Genes associated with the regulation of cell proliferation, apotosis, cellular differentiation and tumor metastasis have a significant expression difference. Two samples of the well and poorly differentiated iced fresh cholangiocarcinoma tissues were analysed by differential display reverse transcription polymerase chain reaction (DDRT-PCR). Thirty-two differentially expressed cDNA fragments were isolated and characterized. Nine cDNA fragments were further identified by reverse Northern blot to be significantly differentially expressed. Three of them belong to the well differential group,named H1、H2 and H3. Six of them belong to the poorly differential group, named L1 to L6. L5 cDNA sequencing result is highly homologous to Laminin Receptor，and L2 cDNA sequence is homologous to many tumor-related expressed sequence tags(ESTs) of Cancer Geome Anatomy Project(CGAP) of National Institute of Health(NIH) (98% identity). The significantly differentially expressed of L2 mRNA level was further identified by reverse Northern blot and RT－PCR among the normal and different differentiated cholangiocarcinoma samples.Sequence 4 (144bp) was cloned which contains the part sequence (120bp) of L2 cDNA according to the designed primers (P1 and P2). Bioinformational methods were used to analyze the sequence of candidate unigene by GOLDKEY Sequence Analyzing Software, and Unigene FXYD6 was found as the homologous gene containing L2 cDNA, and CCR-L2/FXYD6 gene was named for the first time. The genomic sequence of the candidate unigene on Chromosome 11 was found and downloaded by homology searching of L2 cDNA in public databases, and two pairs of primers (P3/P4 and P5/P6) were designed. RT-PCR, molecular cloning, restriction endonuclease digestion, and DNA sequencing technique were performed on total RNA isolated from normal bile duct and cholangiocarcinoma tissues. The expressional difference of CCR-L2/FXYD6 between normal and malignant bile duct tissues was also identified by RT-PCR analysis. A 540bp product was acquired and cloned in pGEM-T plasmid vector. The sequence of the clone(WT5) represents one part of CCR-L2/FXYD6 gene as predicted, which was located on chromosome 11q. So a novel sequence of Unigene CCR-L2/FXYD6 was cloned, and the correlationship between the gene and cholangiocarcinoma was observed, which are high expressed in the cholangiocarcinoma samples and low expressed in normal bile duct tissue. Further study should be performed on the novel gene's function and its relationship with the mechanism of cholangiocarcinogenesis.

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