Biomedical subjects

Dong-Mei Gao

Publications and source records attributed to Dong-Mei Gao.

6 recordsLinked to original sources

PubMed2006

An efficient method of sorting liver stem cells by using immuno-magnetic microbeads.

AIM: To develop a method to isolate liver stem cells fast and efficiently. METHODS: Fetal mouse liver cells were characterized by cell surface antigens (c-Kit and CD45/TER119) using flow cytometry. The candidate liver stem cells were sorted by using immuno-magnetic microbeads and identified by clone-forming culture, RT-PCR and immunofluorescence assays. RESULTS: The c-Kit-(CD45/TER119)- cell population with 97.9% of purity were purified by immuno-magnetic microbeads at one time. The yield of this separation was about 6% of the total sorting cells and the cell viability was above 98%. When cultured in vitro these cells had high clone-forming and self-renewing ability and expressed markers of hepatocytes and bile duct cells. Functionally mature hepatocytes were observed after 21 d of culture. CONCLUSION: This method offers an excellent tool for the enrichment of liver stem cells with high purity and viability, which could be used for further studies. It is fast, efficient, simple and not expensive.

Animals↗

PubMed2005

Molecular cytogenetic characteristics of the human hepatocellular carcinoma cell line HCCLM3 with high metastatic potential: comparative genomic hybridization and multiplex fluorescence in situ hybridization.

The HCCLM3 cell line was established at the authors' institute from the lung metastatic lesions of BALB/c nude mice bearing human hepatocellular carcinoma (HCC) from the metastatic HCC cell line MHCC97-H. It has been shown to have a high potential for lung metastases and extensive metastases when the cells are inoculated subcutaneously or orthotopically in athymic nude mice. In the present study, the molecular cytogenetic characteristics of this cell line were evaluated with conventional G-banding, comparative genomic hybridization, and multiplex fluorescence in situ hybridization. A hyperdiploid karyotype of 53-58 chromosomes with 10 marker chromosomes was identified. The chromosomal aberrations such as i(X)(q10), der(Y)t(Y;18)(q12;p11), der(3)t(3;20) (p25;q13), der(4)t(4;8)(q31;q22)5, der(9)t(9;13)(p21;q22), der(14)t(14;22)(p13;q13), and der(15) t(15;21)(q11;q22) were described for the first time in human HCC cells. The analysis of this cell line through a combination of molecular cytogenetic techniques provides information on the possible molecular mechanisms involved in the metastatic process of HCC.

Animals↗

PubMed2004

Establishment of green fluorescent protein-expressing hepatocellular carcinoma cell lines with different metastatic potential: relevant models for in vivo monitoring of metastasis and angiogenesis.

PURPOSE: To establish stable green fluorescent protein (GFP)-expressing metastatic human hepatocellular carcinoma (HCC) cell lines with different metastatic potential for long-term in vivo studies of metastasis and angiogenesis. METHODS: The pIRES2-EGFP vector, which contains an enhanced GFP gene, was transfected into MHCC97-H and MHCC97-L, HCC cell lines with different metastatic potential. The stability of GFP expression, basic biological characteristics, invasion abilities in vitro, and spontaneous metastasis in vivo of the new cell lines (MHCC97-HG and MHCC97-LG) were studied. Microvessel density (MVD) of orthotopic implanted tumors was compared by anti-CD31 immunohistochemical staining, and real-time angiogenesis and metastasis of GFP-transfected tumors were detected by intravital fluorescent microscope. RESULTS: The GFP-transfected cell lines stably expressed green fluorescence in the absence of G418 over a 36-day period. Compared with the parental cell lines, they exhibited no distinct differences in biological characteristics. MHCC97-HG showed more aggressive invasion and spontaneous metastatic behavior than MHCC97-LG, and even its parental cell line, MHCC97-H (P<0.01). MVD levels induced by MHCC97-HG orthotopic implanted tumors were significantly higher than MHCC97-LG (P<0.01). Real-time angiogenesis and sequential steps of metastasis could be detected clearly under intravital fluorescent microscope. CONCLUSIONS: These two stable GFP-expressing HCC cell lines with the same genetic background and different metastatic potential were established, which could be useful models for monitoring metastasis and angiogenesis of HCC.

Animals↗

PubMed2003

Contributions of lung tissue extracts to invasion and migration of human hepatocellular carcinoma cells with various metastatic potentials.

PURPOSE: The MHCC97 cell line contains two clones with high (MHCC97-H) and low (MHCC97-L) metastatic potentials, for which the pulmonary metastatic rate was 100% vs 40% between the two human hepatocellular carcinoma (HCC) clones. In an effort to elucidate the mechanism of organ-specific metastasis, we studied the effect of lung extracts from C57BL/6 mice on migration and invasion using the MHCC97 cell line. METHODS: Determination of migration and invasion induced by lung extracts to MHCC97 cell lines was examined by chemoinvasion assay. The organization of cytoskeleton was tested using filamentous actin (F-actin) polymerization assay and flow cytometry. The activity of matrix metalloproteinase (MMPs) was analyzed by zymogram. Fluorescence double staining was employed for MMPs and F-actin colocalization in MHCC97-H cells induced by lung extracts. RESULTS: The number of cells in response to extracts of lung, liver, kidney, and spleen in MHCC97-H cells was 64+/-10, 6+/-2, 22+/-4, and 3+/-1, respectively. Of the extracts, lung extracts showed significant differences to promote the migratory and invasive ability for MHCC97-H cells( p<0.001). The number of cells in response to lung extracts was threefold higher in MHCC97-H than that of cells in MHCC97-L ( p<0.001). Confocal laser scan microscope of MHCC97-H cells and MHCC97-L cells stimulated with lung extracts revealed pseudopodia formation around the cell front at the indicated time point. With the time increasing, the pseudopodia formation became increasingly more obvious and distinct. Compared with unstimulated cells, analysis of FACS showed a transient 1.9-fold and 1.7-fold increase in F-actin within 30 s in MHCC97-H cells and MHCC97-L cells, respectively. Confocal laser scan microscopy of MHCC97-H cells stimulated in suspension with lung extracts revealed intense F-actin staining in the periphery of the cells and redistribution of F-actin towards a leading edge. After the cells were incubated with lung extracts, not only expressions of active and latent form of MMP-9 were upregulated, but that of latent form of MMP-2 was increased in the MHCC97-H cells and MHCC97-L cells. The levels of latent and active form of MMP-9(18.8+/-1.2, 100.1+/-1.1), and latent form of MMP-2(22.4+/-1.3) were much higher in MHCC97-H cells than those of MHCC97-L cells, which were 7.8+/-0.3, 40.8+/-2.2, and 8.2+/-0.4, respectively. MMP-9 was mainly localized perinuclear pool when the MHCC97-H cells were incubated with serum-free medium. After the cells were stimulated with lung extracts, MMP-9 were expressed and colocalized with F-actin at the front of extending pseudopodia. CONCLUSIONS: Our results indicate that the expression of matrix metalloproteinase and the pseudopodia formation in MHCC97-H cells may correlate to the metastatic potential; thus, the host environment may contribute to the preferential metastasis of HCC cells to the lung depending on the high level of MMP-9 and migratory ability.

Animals↗

PubMed2003

The abnormalities of chromosome 8 in two hepatocellular carcinoma cell clones with the same genetic background and different metastatic potential.

PURPOSE: Two hepatocellular carcinoma (HCC) cell clones named MHCC97-H and MHCC97-L with different metastatic potential have recently been established from the same parent cell line MHCC97 in our institute. The cytogenetic alterations of these two clones were investigated in this study to explore the possible clues to the mechanism involved in HCC metastasis. METHODS: Their chromosomal aberrations were analyzed with comparative genomic hybridization (CGH), chromosome-specific painting, and two-color fluorescence in situ hybridization (FISH). RESULTS: The aberrations were found in a total of 17 chromosomes, and six kinds of the aberrations including gains of 1q, 7q, 8q, 20, and the losses of 8p23, 21q were found both in the two cell clones and their parent cell line MHCC97. Using modified CGH, with the DNA of MHCC97-L as control to test the MHCC97-H clone, the loss of 8p23 and the gain of 1q31-32, 8q21.3-22, 13q22, 17q22 were highlighted, and the most significant finding was on chromosome 8. Dual color FISH combining a pericentromeric probe and a BAC probe mapping at 8q23.1 was then performed to verify this result, and the signal ratios of the BAC to centromere were 1.43 in MHCC97-H and 1.45 in MHCC97-L, confirming the over-representations at 8q in both cells. Another interesting finding in the dual-color metaphase FISH was the intrachromosomal translocation of 8q to 8p (looked like an isochromosome 8) and non-reciprocal translocation of part of 8q to 4q, which was further clarified and proved by the FISH with whole chromosome 8 painting probe. CONCLUSIONS: The high copies amplification on 8q, the formation of isochromosome 8, non-reciprocal translocation of partial 8q to 4q, and loss of 8p occurred at the same time and are the characteristic chromosomal aberrations of the two cell clones. The chromosome 8p, especially 8p23, might harbor some novel genes related to the HCC metastasis.

Carcinoma, Hepatocellular↗

PubMed1999

Study of angiogenesis induced by metastatic and non-metastatic liver cancer by corneal micropocket model in nude mice.

AIM:To study the angiogenesis induced by liver cancer with different metastatic potentials using corneal micropocket model in nude mice.METHODS: Corneal micropockets were created in nude mice. Tumor tissues and liver tissues were implanted into the corneal micropockets. Angiogenesis was observed usinga digital camera under slit-lamp biomicroscope,and compared among different grafts and incision alone. Vascular responses were recorded in regard to the range, number and length of new blood vessels toward the grafts or incisions.RESULTS:Vascular responses induced by tumor tissues were greater than those by incision alone and liver tissue grafts. LCI-D20 induced more intensive angiogenesis than LCI-D35.CONCLUSION:Highly metastatic liver cancer LCI D20 was more angiogenic than low metastatic cancer LCI D35 and liver tissue. Micropocket was a useful model to study dynamic process of angiogenesis in vivo.

Journal Article↗