| Objective:The growth of tumor can’t depart from the support of nutrients,the phenomenon that nutrient deficiency will occur when the tumor growth to a certain degree.Tumor cells maintain its own growth through angiogenesis,therefore angiogenesis plays an important role in the process of tumor growth.Dependence of multiple myeloma cells on angiogenesis has been recognized.Although a lot of antiangiogenic drugs have some effect but still can’t get away from the phenomenon of resistance.Existing studies have shown that the MM-MVs(myeloma-derived microvesicles)which secreted by multiple myeloma cells in serum starvation stress promote tube formation of endothelial cells.This study suggested that the MM-MVs have a significant role in the process of tumor angiogenesis.We will further study the effect of SD-MVs on the migration ability,tube formation and VEGF secretion level of endothelial cells.The accomplishment of this study will provide a new research mode to the cross-talk mechanism between tumor cells and microenvironment in stress situations,at the same time it will also provide a new strategy to overcome the resistant of antiangiogenesis therapy.Method:Three multiple myeloma cell line were used,KM3,U266 and RPMI8226.The myeloma cells in logarithmic growth were divided into two groups.Normal culture conditions(10%FBS)and serum starvation stress culture conditions(1%FBS)were implemented respectively.After 24 hours,we collected MVs from the normal culture and serum deprivation conditions of KM3,U266,RPMI8226 myeloma cells by classic differential ultracentrifugation,adjust the concentration of MM-MVs,SD MM-MVs with 50μg/ml and co-cultured with endothelial cells.Set MVs from the normal culture and serum deprivation conditions of KM3,U266,RPMI8226 myeloma cells asexperimental group,six groups in total,and take PBS and VEGF(25ng/ml)as control groups.(1)Scratch test: six-well culture plates were used to culture endothelial cells for 12 h,after which they were co-cultured with MVs and SD-MVs in serum-free medium,Lateral migration of endothelial cells were determined after 36 h.The data was presented as scratch area.(2)Transwell migration test : put the endothelial cell suspension in transwell upper chamber,and put MVs,SD-MVs,VEGF or PBS in the lower chamber,co-cultured with endothelial cells for 5h,then fixed and stained.Observe the longitudinal migration level changes of the endothelial cells co-cultured with MVs and SD-MVs by inverted microscope.The numbers of migrated cells in the lower chamber were analyzed.(3)Tube formation test: smear Matrigel in the bottom of 96-well culture plate,and put the endothelial cell suspension in the culture plate,and co-cultured with MVs and SD-MVs 6h.observe the tube formation of MVs and SD-MVs group by inverted microscope.Numbers of closed tube formation were calculated.(4)VEGF levels in the supernate of different groups were tested by ELISA method.Results:1 Scratch test results showed that :there was statistically difference between MVs,SD-MVs experimental group and PBS control group,and the experimental group scratch area was smaller,and SD-MVs had a smaller scratch area than MVs.There was no statistically difference between RPMI8226 MVs,SD-MVs experimental group and VEGF control group.The results stated that MVs promoted HUVEC lateral migration,and compared with MVs,SD-MVs had a more significant lateral migration ability.2 Transwell migration test:there was statistically difference between MVs,SD-MVs experimental group and PBS control group,and the experimental group had more migration cells;and SD-MVs group had more migration cells than MVs group.there was no statistically difference between RPMI8226 SD-MVs experimental group and VEGF control group.The results stated that MVs promoted HUVEC longitudinal migration,and SD-MVs had a more significant longitudinal migration ability.3 Tube formation test showed that there was statistically difference between MVs,SD-MVs experimental group and PBS control group,the MVs and SD-MVs experimental group had more closed tubes,and SD-MVs group promoted more closed tubes than MVs group.There was no statistically difference between RPMI8226 SD-MVs experimental group and VEGF group.The results suggested not only that MVs promoted HUVEC tube formation but also that SD-MVs had a more significant ability of tube formation.4 ELISA showed that there was statistically difference between MVs,SD-MVs experimental group and PBS,the MVs and SD-MVs experimental group secreted more VEGF than PBS group,and SD-MVs group secreted more VEGF than MVs group.Conclusion:1 MM-MVs promote HUVEC abilities of migration and tube formation.2 SD-MVs have a more significant effect on promoting angiogenesis than MVs.3 HUVEC co-cultured with MM-MVs secret a higher level of VEGF,and SD-MVs group secret a higher level of VEGF,compared with MVs group. |
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