| Objective: Vesculogenic mimicry is a special blood supply of highly aggressive tumor.This research isolated tumor stem-cell-like cells form epithelial tumor of human ovary(or ovarian tumor)through serum-free stem cell medium cultured.By Flow cytom etry,Flat colony formation test and tumor formation test in vivo of nude mice to identify the characteristic of tumor stem-cell-like of isolated cells.Compared the ability of VM formation between selected cells and maternal cells,and then tested the expression levels of Metalloproteinases-2(MMP-2)and Metalloproteinases-9(MMP-9)in both of them.Methods:1 Serum-free supension cultureThe maternal HO8910 cells were cultured in serum-free stem cells medium included mutiple cell growth factors by continuous passage,observe the changes of suspension cell spheres along with passages,took photos and record,and gathered 1st,3rd,5th,7th generations suspension cell spheres for subsequent experiment.2 Flow cytom etry tested the expression levels of CD133 positive cells in the 1st,3rd,5th,7th generations suspension cell spheresEach generation was labeled by CD133 antibody,by Flow cytom etry tested,these cells expressed strong fluorescence were CD133 positive cells,these cells expressed feeble fluorescence were CD133 negative cells,and calculated the rate of CD133 positive cells in each generation.This experiment was repeated to 3 times.3 The experiment of Flat colony formation test in vitroCompared the colony-forming efficiency between the 7th suspension cells and maternal HO8910 cells.The single cell suspension of this two kind of cells prepared,and seeded them in the culture box and cultured for 1-2 weeks in an incubator.This experiment was repeated to 3 times.4 The experiment of tumor formation in vivo of nude micePreparing the single cell suspension of the 7th suspension cells and maternal HO8910 cells,Counting the number of the 7th suspension cells were 1×103 and the maternal cells were 1×105,and respectively resuspended into 0.2ml PBS.Maternal cells inoculated the right thigh subcutaneous and the 7th suspension cells inoculated the left thigh subcutaneous of nude mice,all six female nude mice,observed the tumorigenesis situation in nude mice subcutaneous every day.5 Three dimensional cell culture to observed the formation of VMThe 7th suspension cells and maternal HO8910 cells were made into single cell suspension and respectively inoculated into Matrigel which three dimensional cell culture on it.Observed the formation situation of VM in the same time interval by inverted phase contrast microscope,and randomly selecting five views of upper,lower,left,right,and center took photos and record.This experiment was repeated to 3 times.6 Real-time PCRThe expression levels of Metalloproteinases-2 and Metalloproteinases-9 of the 7th suspension cells and maternal cells tested by RT-PCR,This experiment was repeated to 3 times.7 Statistical analysisA Statistical software(SPSS 17.0)was used for statistical analysis.Measurement dates were expressed as mean ± standard diviation,Compared two-groups used t-test.Inspective level was assigned as ?=0.05,when P<0.05,there was statistically significant;when P>0.05,the difference was no statistically significant.Results:1 Suspension culture of the maternal HO8910 cellsThe maternal HO8910 cells were cultured in serum-free stem cell medium,after 24 hours most of cells adhered on the bottom of the culture flask,while the others were state of suspension.With other 24 hours culture,most of adherent cells were dead,while suspension cells obviously increased,the suspension spheres only consist of a few cells.All adherent cells were break down when cultured 4-6 days,while the number and size of suspension cells spheres increased markedly as the cell repeatedly passages.We observed the 1st suspension cell spheres only had a small number of cells,with similar size and some refraction,the tight connection between these cells but can distinguish the boundary of cells.The 7th suspension cell spheres had dozens cells circled,refraction increased and third dimension full,with so tightly connection between cells that we can not clear the boundary,the size and shape of cell still keep similar to the first suspension cell spheres.The results illustrated suspension cells have a capable of self-renewal.2 The result of Flow cytom etryThe results of flow cytom etry illustrated that the expression level of CD133 positive cells in the 1st,3rd,5th,7th suspension cell spheres were increased,the rate of positive expression is 0.23%,31.74%,79.25%,88.18% respectively.3 The result of flat colony formation test in vitroFlat colony formation test indicated that the colony-forming efficiency of the 7th suspension cells is 45.67±1.53% and maternal HO8910 cells is only 11.00±2.00%,the former is the latter's 4 times,there was statistically significant(t=23.859,P<0.00).4 The analysis of tumor formation in vivo of nude miceThe 7th suspension cells(1×10~3)inoculated the right thigh subcutaneous,observed six weeks,the all six mice formed visual transplantable tumor(6/6).The maternal cells(1×10~5)inoculated the left thigh subcutaneous,observed six weeks,without any mice formed visual transplantable tumor(0/6).5 The ability to form VM in three dimensional cell culture modelUnder the condition of Matrigel which three dimension cell culture on it,the 7th suspension cells appeared connection of cells after 2 hours,the typical latticed structure formed after 4 hours,loosened and faded after 48 hours.The connection of cells is tightly and isscross when 4 hours.The maternal cells appeared connection of cells after 4 hours,but the connection is loose and irregular.The connection of cells reached most after 8 hours,but the typical VM structure is seldom,loosened and faded after 24 hours.6 The expression situation of MMP-2,MMP-9 tested by RT-PCRThe results of RT-PCR revealed: the expression levels of MMP-2 in the 7th suspension cells is 6.0 times the maternal cells,the difference was statistically significant(t=-4.544,P=0.045).the expression levels of MMP-9 in the 7th suspension cells is 6.7 times the maternal cells,the difference also was statistically significant(t=-4.019,P=0.014).Conclusion:1 The maternal HO8910 cells were cultured in serum-free stem cells medium included mutiple cell growth factors can continuous passage and got suspension cell spheres,it is identified that the 7th generations suspension cell spheres could express higher tumor stem cell marker CD133,had stronger colony-forming efficiency and tumor formation ability in vivo of nude mice,those revealed that the 7th suspension cell spheres have the characteristic of tumor stem cells.2 The 7th suspension cell spheres have a stronger ability to form VM than maternal cells,and high-expression MMPs.It's speculation that tumor stem-cell-like cells may express higher MMPs to promote the formation of VM by inducting the remodeling of extracellular matrix and tumor cells,provide blood supply to the growth and metastasis of tumor.It's helpful to realization the mechanism of the growth,metastasis and recurrence of ovarian cancer. |
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