| Objective:Vasculogenic mimicry is a microcirculation structure of highly aggressive tumor cells for self-blood. This paper researches the isolation and culture of the three different patterns of single cell cloning by using human epithelial ovarian cancer cell line SKOV3. Comparing their capacity of tumorigenic in vivo and in vitro and computing the rate of colony forming efficiency. To observe the formation ability of VM and detect the expression levels of tumor stem cell marker CD133 and matrix metalloproteinase MM-2 and MMP-9.Methods:1 SKOV3 single cell cultured into three forms of single cell clone with limited dilution method.Selected SKOV3 cells in Logarithmic growth phase and diluted continuous multiple very low density by the principle of gradient dilution multiple(containing 0.5-1 cells in each 96 well plate). The next day, selected and labeled the hole with only one cell. The hole formed large clones after cultured for 10-14 days. Distinguished and labeled different cell clones of different forms. And cultured these cells continuely for Subsequent experiments. The experiment was repeated to 3 times.2 The experiment of tumorigenicity in vivo30 female nude mice(BALB/c-nu) were divided randomly into three groups and inoculated with three different types of cells. Under sterile conditions, counting the number of the holoclone cell of 1000, meroclone cells and paraclone cells of 1x105, and resuspended into 0.2ml PBS, and inoculated in right thigh subcutaneous of nude. Observe of the tumorigenesis situation in nude mouse skin. The experiment was repeated to 3 times.3 The experiment of soft agar colony formationPreparing of single cell suspension, and let the holoclone, meroclone, paraclone were seeded in the soft agar culture box, and cultured for 2~3 weeks. Microscopic counting clones containing at least 50 cells, and calculated the CFE. The experiment was repeated to 3 times.4 Three-dimensional cell cultureHoloclones cells, meroclones and paraclones cells were inoculated into Matrigel that 3D cell culture on it. Selecting randomly 5 views of upper, lower, left, right and center by an inverted phase contrast microscope. Observed the formation of VM in the same time interval, and took pictures and record. The experiment was repeated to 3 times.5 The expression level of CD133 and MMP-2 and MMP-9 by Western Blot method. The experiment was repeated to 3 times.Results:1 Screening of monoclonal cellsThe SKOV3 cells were seeded in 96 holes plate by using limited dilution method. Selected and marked the hole contains only 1 cell after inoculation of 24 h. To distinguish the morphology and classification and count the cell clones after culture 10-14d(this part of the experiment were confirmed by two people). Holoclone cells were round or oval. The cell arranged concentratedly and neatly, morphological rules. The cell in Holoclone has the advantages of small size, less cytoplasm and no obvious pseudopodia or protuberance. Paraclone cells were irregular shape, loose, large volume. The cell in Paraclone had abundant cytoplasm and obvious pseudopodia or protuberance. The morphology of Meroclone was located between the holoclone and paraclone, not rule as holoclone. The cell in Meroclone arranged relatively uniform, smaller volume. The arrangement of cells tended to be loose, the surrounding cells appeared pseudopodia. The average ratio of the experiment was repeated 3 times to calculate the three kinds of cloning: holoclone is 17.4±0.8%, meroclone is 30.1±0.4%, paraclone is 52.5±1.1%. Under the same conditions, holoclone had rapid proliferation, but meroclone and paraclone disappeared partly on the observation of 10 weeks. Holoclone still maintained rapid growth on the observation of 12 weeks, and the small part of all the remaining meroclone and paraclone occurred apoptosis or could not continue passage. The morphology of holoclone on the observation of 16 weeks was similar to maternal SKOV3 cells, logarithmic growth. Subsequent experiments were performed using the cells cultured for 12 weeks ago.2 Analysis of ability in vivo tumorigenic1000 holoclone cells were seeded in female nude mice subcutaneous thigh. The tumor formation rate was 80% on the observation of 7-10 days.1×105 meroclone cells were seeded in female nude mice subcutaneous thigh. The tumor formation rate was 0 on the observation of 6 weeks.1×105 paraclone cells were seeded in female nude mice subcutaneous thigh. The tumor formation rate was 0 on the observation of 6 weeks.3 Rate of Clone formingSoft agar colony formation test results: The holoclone cells had a CFE of 16.7±3.8% and meroclone cells had a CFE of 6.5±1.5%, but the paraclone cells had a lower CFE of 1.2±0.7%(P <0.05). CFE of holoclone was two times to ten times than meroclone and paraclone.4 The ability of three kinds of single cell clone formation VMIn 3D cell culture conditions, holoclone had the formation of typical VM tubular like structures in the 6 h, peaked at 12 h and began to loose after 72 h. There was a lack of VM formation in paraclone and meroclone.5 Expression of CD133 and MMP-2, MMP-9 by western blotThe expression level of CD133 protein in Holoclone group was significantly higher than that of the maternal SKOV3 group, while also higher than the meroclone and paraclone group(P <0.05 F=33.524). The expression level of MMP-2 protein in Holoclone group was significantly higher than that of the maternal SKOV3 group, while also higher than the meroclone and paraclone group(P <0.05 F=11.641). The expression level of MMP-9 protein in Holoclone group was significantly higher than that of the maternal SKOV3 group, while also higher than the meroclone and paraclone group(P <0.05 F=14.336), the difference was statistically significant.Conclusion:1 Single cell cloning on human ovarian cancer cell line SKOV3 has heterogeneous in morphology and differentiation, and holoclone cells has the characteristic of tumor stem-like cells.2 Holoclone is a SKOV3 cell subset of the higher ability of invasion and recurrence. Induction of extracellular matrix remodeling can form VM through its plasticity and MMPs, provide adequate blood supply to the invasion and metastasis of ovarian malignant tumor. It is helpful to understand the mechanism of the invasion and recurrence of ovarian cancer. |
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