| Objective:To observe the influence of hepatocarcinoma stem-like cells on the overall survival of NOD/SCID mice, and to study the possible causes of this phenomenon in vivo and in vitro.Methods:1. SK-Hep-1cell was cultivated in serum-free medium(SFM) supplemented with growth factors and cancer stem-like cells reforming into floating spheres were isolated.2. Cell proliferation and cell cycle of spheroid cells were detected by MTS and Flow Cytometry.3. Colony formation assay was conducted to identify the self-renewal and propagation abilities of both spheroid cells and SK-Hep-1cells in vitro. Tumor formation assay was conducted to identify tumorigenicity of both spheroid cells and SK-Hep-1cells in NOD/SCID mice.4. MTS were conduced to detect drug resistance of spheroid cells treated with5-Fu, DDP, ADM and NVB.5. The mRNA and protein levels of Oct4, Nanog, BMI-1, Notchl and SMO in spheroid cells were detected by Real-time PCR and Western Blot.6. HSC was implanted subcutaneously in the NOD/SCID mice to observe its influence on the mice’ overall survival.7. Observe the organs’ structural changes of NOD/SCID mice under microscope after HE dyeing. Detect the influences of HSC on the NOD/SCID mice’ blood biochemical indicators. Observe hepatocyte apoptosis and proliferation effects of NOD/SCID mice applying Tunel and PCNA staining after HSC was implanted.8. Collagenase digestion method was applied to separate the mice primary liver cells. 9. Flow Cytometry combined with CFSE dyeing were applied to detect the proliferation effects of HSC on the human and mice normal liver cells.10. Flow Cytometry combined with AnnexinV/PI dyeing were applied to detect the apoptosis effects of HSC on the human and mice normal liver cells.11. Flow Cytometry combined with PI dyeing were applied to detect the cell-cycle blocking effects of HSC on the human and mice normal liver cells.Results:1. Human hepatocarcinoma SK-Hep-1cell could be maintained in serum-free medium with floating-culture methods with successive generations.2. Most spheroid cells were in low growth kinetics. Most of them were in silent period, which is Go/G1phase of cell cycle.3. Spheroid cells exhibited the colony formation capability. Colony formation efficiency of spheroid cells (51.63%) was much higher than SK-Hep-1cells (17.54%). HSC harbored a high property of tumorigenicity,1000SCs generated tumor nodules in the NOD/SCID mice and the formed tumor nodules displayed similar histology to that of the original tumor.4. After treated with a certain concentration of chemotherapeutics for48hrs, spheroid cells are found to be more viable when compared to matched SK-Hep-1counterparts.5. Spheroid cells highly expressed mRNA and protein levels of Oct4, Nanog, BMI-1, Notchl and SMO compared with SK-Hep-1cells.6. The overall survival of NOD/SCID mice inoculated with HSC was significantly shortened compared to SK-Hep-1group according to survival analysis.7. The albumin levels were significantly decreased and the ALT levels significantly increased of the HSC group mice when compared with SK-Hep-1group. Observed the liver tissue of HSC group mice, the hepatic cord was in disorder, liver cells showed vacuolar degeneration, eosinophilic change and small focal necrosis, sinusoidal expanded and mononuclear macrophages was in severe hyperplasia, the condition was worse compared with SK-Hep-1group. Tunel staining showed the apoptotic cells of HSC group in liver tissue was significantly increased, PCNA staining showed the PCNA-positive cells in HSC group was much fewer than that of the SK-Hep-1group.8. Mice primary hepatocytes were bulky with irregular or polygon morphology, single or dual-core, clear cell-to-cell boundaries.9. Liver cell proliferation was significantly inhibited when co-cultured with HSC-cultured supernatant and HSC compared with SK-Hep-1.10. Liver cell apoptosis was significantly induced when co-cultured with HSC-cultured supernatant compared with SK-Hep-1. Co-cultured with HSC and SK-Hep-1has no effect on the liver cell apoptosis.11. The liver cells percentage of S phase increased when co-cultured with HSC-cultured supernatant and HSC, while cells percentage of Go/G1, G2/M decreased. Compared with SK-Hep-1, HSC showed a significant cell cycle blocking.Conclusion:Using serum-free medium with floating-culture methods, we isolated the hepatocarcinoma stem/progenitor cells subset from the human hepatoma SK-Hep-1cell line. HSC performanced a stronger liver injury role, induced liver cell apoptosis and inhibited the regeneration of liver cell in vivo. At the same time we further indicated that HSC could inhibit proliferation of normal liver cells, induce liver cell apoptosis and cause cell cycle arrest. From the results above, we concluded that HSC induced hepatocytes apoptosis, caused mice liver injury and inhibited hepatocytes regeneration and liver restoration. This might be one of the reasons which leaded to increased mortality of tumor-bearing mice. |
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