The Study On The Effects Of Exosomes Derived From Bone Marrow Stromal Cells On The Drug Resistance Of Leukemic Cells

Background:Acute lymphoblastic leukemia (ALL) is the most common type of hematologic malignancies. Recent years, lots of improvements have been made in treatment of ALL. However, there are still some patients with poor outcome or relapsing after remission. Drug resistance is one of the most vital causes for this phenomenon. Exosomes are nanoparticles secreted by viable cells, which play an important role in development and progression of hematologic diseases. It has become an emerging researching field.Objective:1. To isolate exosomes from BMSCs-conditioned medium and confirm them by morphology and surface markers.2. To explore the effects of exosomes on the proliferation, apoptosis and cell cycle of Nalm-6 cells and the mechanisms how exosomes paly their role in these actions by co-culturing the Nalm-6 cells with exosomes.Methods:1. Culturing the BMSCs with serum-free medium, isolating exosomes from BMSCs-conditioned medium using differential centrifuge method, and confirming exosomes by transmission electron microscope and western blot respectively.2. Detecting the effect of BMSCs-exosomes on the proliferation of Nalm-6 cells using CCK-8 method.3. Examining the effect of BMSCs-exosomes on the apoptosis of Nalm-6 cells using flow cymetry.4. Determining the effect of BMSCs-exosomes on the cell cycle of Nalm-6 cells using flow cymetry.5. Detecting the effect of BMSCs-exosomes on the expression levels of apoptotic-related proteins in Nalm-6 cells by western blot method.Results:1. Exosomes were successfully isolated from BMSCs-conditioned medium. The size and shape of the isolated exosomes were confirmed using transmission electron microscopy, and typical cup-shaped membrane particles with a diameter of 50 nm to 150 nm were observed. Western blot analysis showed that exosomal markers CD63 and HSP70 were detected in these exosomes.2. BMSCs-exosomes can promote the proliferation, inhibit the apoptosis and promote the cell cycle of Nalm-6 cells, thus influence the drug resistance of leukemic cells.3. BMSCs-exosomes could inhibit the down-regulation of BCL-2, full-length caspase-3 and full-length PARP in Nalm-6 cells induced by VP16; and decrease the expression levels of BAX, cleaved caspase-3 and cleaved PARP.Conclusion:1. Exosomes can be successfully isolated from BMSCs-conditioned medium and contain various kinds of miRNAs.2. BMSCs-exosomes may induce drug resistance of leukemic cells through regulation of the cell proliferation, apoptosis and cell cycle.