| Hepatocelluar carcinoma (HCC) is one of the most common human cancers worldwide, and metastatic recurrence is the major obstacle to improve the prognosis of HCC patients. In order to known how the accumulation of genetic changes give rise to this aggressive phenotype, consistent efforts have been made by us to get the ideal cell model for in vitro studies on mechanisms underlying the carcinogenesis and tumor progression of HCC. Recently, two cell clones (MHCC97-H and MHCC97-L) with different metastatic potentials were successfully subcloned from the metastatic human HCC cell line MHCC97 in our institute, and another cell line designated HCCLM3 which was able to produce more extensive metastases via both subcutaneous and orthotopic inoculation in athymic nude mice was successively established. These cells provide us valuable tools for identifying cytogenetic aberrations and molecular markers associated with the metastasis of HCC.In our previous studies, through CGH analysis of both clinical tumor samples and the animal models, 8p deletion was found to be one of the most obvious aberrations in HCC, and might be associated with HCC metastasis. In most recently, we had used more precise genome-wide scan methods such as microsatellite analysis to detected about 22 HCC tumor and their matched metastasis focal, and the 8p23.3 and 8p11.2 were found to be related to progression and metastasis of HCC.To identify whether our previous founding were exist in the series of our newly established cell cultures with the same genetic background and different metastatic potentials, and to make clear what are congenerous and various traits in the chromosomal or DMA sequence levels between these cells, in this study, a combination of conventional G-banding, comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and arm or locus-specific fluorescence in situ hybridization (FISH) were used to comprehensively characterize molecular cytogenetics aberrations of the above cell cultures. This will provide clues to the molecular mechanisms involved in the HCC metastasis.Part oneEstablishment of the FISH-based molecular cytogenetic techniques and its primary applicationThe major objectives of this part were to establish the novel methods based on fluorescence in situ hybridization (FISH) techniques such as comparative genomic hybridization (CGH), multiplex fluorescence in situ hybridization (M-FISH) and whole chromosome painting (WCP) in our institute.First, indirect two-color FISH was performed on the metaphase of MHCC97-H cells. The alpha satellite pericentromeric probe specific to chromosome 8 (D8Z2) was obtained from Oncor (Qbiogene, Cedex, France), and the DMA of BAG clone RP11-328M16 mapping at 8q23.1 was extracted and labeled by ourselves. The results showed that the penetrating ability, specificity and signal strength of the both probes were acceptable. The trisomy of chromosome 8 and the non-reciprocal translocation of partial 8q to one chromosome in group B (Chromosome 4) were found.Second, the directly labeled whole chromosome painting (WCP) probes were purchased from Vysis Company (IL, USA, the MFISH probes also came from this incorporation), and the steps of the hybridization (almost the same as MFISH) were successfully optimized. The findings of numerical and structural changes of chromosome 8 in MHCC97-H were confirmed.Based on the experience of two-color FISH, we analyzed human hepatocellular carcinoma (HCC) cell line SMMC-7721 with low or none metastatic potential by Indirect CGH. The key parameters of CGH, such as the signal granulation, signal intensity, chromosome length/width and separated chromosomes were all reached the default value. The gains of 1p31, 1q25, 3p22-pter, 5p, 6p21.3-pter, 7p13-pter, 8p23, 8p11-q12, 9q22-qter, 10p12-q21, 11pter-q21, 11p12-q14, 14q13-qter, 15q15-qter, 17p, Xp14-pter and the losses of 4q31-35, 9p21-3, 13q21-31, 18q,Yq were detected in this cell line. These changes may serve as the control for our subsequent research on mechanism o...
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