| Aims: Hepatocellular carcinoma (HCC) is a kind of malignant disease that is difficult to treat and has a high incidence and a high mortality at present. The established cell lines and animal models of human liver cancer have settled a good foundation for study of pathogenesis and treatment of HCC. There are variations of chromosomes and genie location among HCC cell lines, and the established HCC cell lines in vitro could be transformed, include loss and drift of genes, etc, also there is obvious difference between cell lines and their original tissue. Yet the same cell line could be transformed after passaged repeatedly, so the test results are uncertainty. Clinic and laboratory data shows limit resource of cell lines can't satisfy the needs of study. More kinds of new hum-an HCC cell lines are needed, and can be help to study the commonand different points of pathogenesis, progress and metastasis ofliver cancer. Drug sensitivity can be tested through kinds of HCCcell lines, so that some efficient drugs can be screened againstcertain liver cancer and provide instruction on clincic treatment.Methods:1.Establishment of human HCC cell lineThe sample of human HCC was primary cultured by tissue culturing. The single HCC cell were obtained by natural purification and anchoring repeatedly. The cells were passaged with trypsin. Growth status was observed under light microscope.The cells were freezed with temperature down slowly in liquid nitrogen and resuscitated quickly at 37C. 2.Characteristics of human HCC cell line FHCC-98.FHCC-98 cell morphology was observed under light and electron microscope. The survival rate was measured by counting cells stained with protamine Blue.Seeding efficiency was measured by counting cell number every 2 h. Cell growth curve was plotted with MTT assay. Studies of cell cycle and stage were performed by flow cytometry.Chromosomes was made by traditional method and analyzed by photographed. Cloning efficiency was measured by counting clone number growing in soft agar. The coagulation characteristics was detected by ConA test. Expressions of tumor markers such as AFP, CK and HAbl8G/CD147 were detected by SP immunocytochemistry or Western blotting. LDH isoenzymes were detected by polyacrylamid gel PAGE and photographed analyzed. Xenograft was performed by inoculating FHCC-98 cellsinto the flanks of the nude mice s.c and the tumors were observed, thus xenograft rate was counted. Cell contamination was detected by transmission electron microscopy.3.Study of drug sensitivity against HCC cell lines3.1 MTT assay was used to detect the inhibition rate of HCC cell growthTaxotere and ADM were used to effect on FHCC-98 and MHCC-97-L cells. OD was measured by ELISA at different time and concentration, and inhibition rate was counted.3.2 Invasion test was used to investigate the mobility inhibition of FHCC-98Millicell-PCF transwell chambers were put in 24-plate, taxotere and ADM were added to test group, and no drugs in controlled one. Transwellchamber's membrane was HE stained and photographed. Inhibition rate was measured with imaging analyser.3.3 DNA damage of FHCC-98 cell was dectected by SCGETwo layers of gel was made, and FHCC-98 cells were el-ectrophoresised in alkaline condition after lysis, then nucleus damage by taxotere and ADM was observed under fluorescence microscope. Results:1. Cells grew instably at the early stage of primary culture and needed a high density when passaged. Population doubling time was not the same. But the cells grew very stably at 12th passage. Cells were mainly polygonal epithelial-like in various size over 95%. Cells grew in overlap and piled up in high density (>1 X 106/mL). Passaging time was 2 -3 days average.2. Transmission electron-microscopy showed cells were invarious size, mainly irregular type. There were abundant microvilli on the cell surface. Desmosomes and gap junction could be seen between cells, also with a few junctional complexs. There was a tendency to form structure of gland and strand alike. St...
|
PDF Full Text Request