Preliminary Studies Of Mouse Embryonic Stem Cells And Melanoma B16Cells Interaction In Vitro

Malignant tumor is the uncontrolled proliferation anddysdifferentiation of cells.Biological behaviors of tumor cells and thelife process of early embryonic development are very similar. Metastaticcancer cells similar to stem cells.They have shown the self-renewal andsimilar cell molecular markers, showing the two types of cells have abasic plasticity.If the two cells,very similar,were placed in co-culture syetem,thatwhat would be the phenomenon? Who would dominant,or who wouldhave a more impact on the other and what effects would be?In thissubject,we intended to establish C57BL/6mouse embryonic stem celllines,to focuse on the co-culture of mouse embryonic stem cells andmouse malignant melanoma B16cells,to explore the two cellsinteraction between embryonic stem cells and tumor cells in co-culturesyetem and the influence on the biological behavior of tumor cellsco-cultured with embryonic stem cells.objective 1.To optimize mouse embryonic fibroblast feeder layers, create astable and efficient C57BL/6mouse embryonic fibroblast feeder layersculture system, for the purification of mouse embryonic stem cells andestablishment of Cell lines.2.To establish a stable C57BL/6mouse embryonic stem cell lines.3.To establish mouse embryonic stem cells and B16cellsco-culture system in vitro,detect changes of the part biological behaviorsof B16cells co-cultured in vitro and preliminary study interaction ofmouse embryonic stem cells and B16cells in vitro and its mechanism.Methods1.Taking pregnancy12.5-14.5d fetal rat,using tissue digestionmethod mouse fibroblast cells were isolated and cultured.MTT assaydetermined MEFs proliferation,the optional concentration of mitomycinC and the time treating with mitomycin C on MEFs、cells algebras andthe optional planting density of cells in preparation of feeder layer.2.Mouse blastocysts gestation3.5days were placed on feeder layercell culture systems seeded different densitiy. Observeing the growthstatus of the blastocyst、inner cell mass and ES cells, further to validatethe MEF feeder layer were used for isolation and culture of ES cellsresults.3.C57BL/6mouse3.5days blastocysts collected were placed on the mouse fibroblast feeder layer incubation.After3-5days the inner cellmass were separated、amplified and passaged over35generations.EScolony growth was observed.ES cell colonies were identified throughmorphology、 alkaline phosphatase staining、 early embryo-specificantigen(SSEA-1) and OCT4staining,and differentiation experimentsin vivo.4.Under the four different conditions, mouse embryonic stem cellsand B16cells co-cultured,in order to select the right mouse embryonicstem cells and B16cells co-culture system.5.Through mouse embryonic stem cells and tumor cells in vitroco-culture model,Effects of mouse embryonic stem cell on morphologyand growth behavior of tumor cells were observed.MTT assaydetermined the changes of co-cultured B16cells proliferation andadhesion ability. Transwell small chamber method determined thechanges of co-cultured B16cells invasion and migration ability.Results1.Mouse fibroblasts dealed with mitomycin C10micrograms permilliliter concentration、3-3.5hours,20micrograms per milliliterconcentration、2-2.5hours,were inhibited better. The viability of mousefibroblasts seeded each well (1-2)×104cells in96-well plates weremore stable than thoes (3-6)×104cells.The viability of mouse fibroblasts dealed with mitomycin C,1to4-generation were more stablethan5,6-generation,and the vitality could maintain a longer time.2.Under different inoculation density feeder layer cell culturesystems, blastocyst adherence rate and hatching rate of the inner cellmass and formation rate of embryonic stem cell clones wereanalyzed.Blastocyst adherence rate and hatching rate of the inner cellmass and formation rate of embryonic stem cell clones were all higheron feeder layer cells prepared under optimization ofconditions.Ultimately,ES cell colony that could passage stably could beisolated and cultured.3.ES cells isolated and cultured could stably passage to more than35generations, and showed a colony-like growth.AKP staining,immunohistochemistry of SSEA-1surface antigen and OCT4antigenwere positive.Inoculated in nude mices and C57BL/6mices could formteratomas,witch had three germ layers tissue components in H&Estained. These were in line with the characteristics of mouse ES cells.4.Tumor cells before vaccination,after the embryonic stem cellsjoined,and the embryonic stem cells before vaccination,after tumorcells joined,and two cell simultaneous administration conductedco-culture with ES conditioned medium;Tumor cells beforevaccination,after the embryonic stem cells joined conducted co-culture with ES conditioned medium DMEM/F12+20%fetal bovine serummedium,in order to select the right mouse embryonic stem cells and B16cells co-culture system.5.In mouse embryonic stem cells and B16cells co-culturesystem,mouse embryonic stem cells were able to invade tumor cells andform their own growth space.Compared with the controlgroup,co-cultured tumor cell proliferation,adhesion,invasion andmigration ability were significantly lower.ConclusionMouse embryonic fibroblast feeder layer prepared under theoptimized conditions,could better support the blastocyst adherent、innercell mass hatching out and embryonic stem cell colony formation andgrowth.The embryonic stem cells obtained were identified in accordancewith the general characteristics of mouse embryonic stem cells.Steadilyspreaded to more than35generations remained embryonic stem cells stemcharacteristics.Ultimately,the stability C57BL/6mouse embryonic stemcell lines were established.Using a co-culture experimental model,mouseembryonic stem cells and B16cells co-cultured in vitro.Mouse embryonicstem cells were able to invade tumor cells and form their own growthspace,and lower their proliferation、adhesion、invasion and migrationability in vitro co-culture system.