Effects Of Space Environment On Biological Characteristics Of Melanoma B16-F10 Cells

Objective:1.To examine the similarities and differences of the biological character -istics(cell morphology,proliferation curve,cell cycle,tumorigenesis,etc.) of the subcloned cell lines and the parental wild-type B16-F10 cells,the subcloned cell lines were obtained by monoclonal of the wild-type B16-F10 cells.2.Studying the effects of the space environment on biological characteristics of melanoma B16-F10 cells by comparing with the results of the space environ -ment mutagenesis.Methods:The wild-type B16-F10 cells were monocloned by limited dilution method,10 subcloned cell lines were selected at random from a total of 104 subcloned cell lines as the experimental group.The serial number is 2 #,3 #,9 #,32 #,45 #,50 #,69 #,74 #,83 # and 99 #,the parental wild-type B16-F10 cells as the control group.1.In vitroThe morphological changes were examined with the phase contrast microscope.The 3(4,5dimethylthiazol2yl)2,5diphenyltetrazolium bromide,(MTT)assay were car -ried out to evaluate the cell proliferation.FCM(Flow Cytometry) was used to detect the changes of cell cycle.2.In vivo The control group and experimental group B16-F10 cells were inoculated in C57BL /6J mice,each kind of cell was injected into twenty mice which had been divided into two groups randomly,each group had ten mice,one group were inoculated into the abdominal cavities to observe the survival times of tumor mice,ano -ther group were inoculated under the chest skins to record the time named as the forming-tumor time when the diameters of tumors exceeded 3mm.The tumor mice were killed when cells had been inoculated 2 weeks later,then,tumor ti -ssue,thymus and spleen were ablated.The tumor tissues were weighted to ob -serve the forming-tumor abilities of B16-F10 cells,the thymus and spleen were weighted,then calculated thymus factor and spleen factor,to observe the ch -anges in the immune system of mice,the melanoma tumor tissues were examined histopathologically by the use of HE Chahe concept staining.3.Analyzing the results and comparing with the results of the space mutag -enesis.Results:1.In vitroUnder the optics inverted microscope,the cell line distribution of the control group and the experimental group were even,mostly presented anomalous. Compared with the control group,the cell line distribution of the experimental group had no significant difference.MTT test results indicated that compared with the control group,the proliferation speed of subcloned cell lines showed multiple variation.09 #,32 # cell lines were faster than the control group, 69 #,02 # and 50 # cell lines were slower than the control group,other cell lines had no significant difference compared with the control group.The FCM test results indicated:compared with the control group,G0/G1 time cell of 32# cell line reduced not obviously,but G0/G1 time cell of 09# cell line reduced obviously,S time cell increased,G0/G1 time cell of 69#,02# and 50# cell line increased obviously,G2/M time cell,S time cell reduced,were consistent with each correspondence cell multiplication speed tendency.2.In vivoCompared with the control group,the latent period of tumor formation and the survival time of 69 #,50 #,02 #,83 # cell lines were prolonged,tumor weight were significantly reduced,spleen coefficient were obviously increased,09 #,32 # cell lines had a marked increased in tumor weight,the survival time were shortened obviously,thymic coefficient and spleen coefficient were reduced obviously,other cell lines compared with the control group there were no significant difference.HE staining of tumor tissue showed that:the control group and 32# cell lines have more tumor necrosis organization,69# and 50# cell lines had less.3.Selected one cell line which showed that the proliferation speed slowed down obviously,the latent period of tumor formation and the survival time were prolonged,the tumour weight were reduced,the serial number is 69 #.The cell line whose tumorigenesis was weakened and space mutagenesis gained the cell line whose immunogenicity was strengthened were compared,studying the effects of the space environment on biological characteristics of melanoma B16-F10 cells.4.Compared with the control group 69# cell line,the shape of the space group 11# cell line had large changes,cell volume were increased,protuberance increased.MTT measure results revealed that the proliferation speed of the space group 11 # cell line slowed down obviously.In vivo experimental results revealed tumor weight of the space group 11# cell line were significantly reduced,the survival time were prolonged.Tumour organization slice H.E dye results revealed in the tumor organization of the space group 11 # cell line tumor-infiltrating lymphocytes can be seen.Conclusion:1.Compared with the parental B16-F10 cells,the biological properties of the subcloned cell lines(cell shape,proliferation curve,cell cycle,tumorige -nesis,etc.) showed a multiple variation.2.Compared with the control group 69# cell line,the space environment have the stronger mutagenesis function to the biological properties of melanoma B16-F10 cells.