| Object: Secreted by most cells in the body,exosomes are nanoscale vesicle-like bodies that carry a variety of bioactive molecules,such as proteins and nucleic acids,between adjacent cells and distal cells.It has been well shown that exosomes mediate the transduction of biological signals and consequently change the functional state of recipient cells.Methods:1.Extraction and identification of exosomes from cells:(1)Human intestinal epithelial cell lines NCM460,colon cancer cell lines SW620 and SW480 were cultured,and exosomes were isolated by hypercentrifugation.(2)Nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM)were used to identify the particle size and morphology of exosomes.Western blotting was employed to detect the expression of exosome markers Alix,TSG101 and CD63.2.Effects of exosomes from cells on SW480:(1)The isolated exosomes were labeled with fluorescent dye Di I,and then co-cultured with SW480 cells.The uptake of exosomes was observed under a confocal microscope.(2)Transwell assay was performed to detect the effect of exosomes on the migration and invasion ability of SW480 cells.(3)Western blotting was conducted to analyze the influence of exosomes on related signaling pathways.3.Extraction and identification of plasma exosomes:(1)Plasma exosomes were separated by ultracentrifugation,precipitation and Size-exclusion chromatography(SEC).Three types of SEC were Sepharose CL-2B,Sepharose CL-4B and QEV columns.(2)By using Nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM),the particle size and morphology of exosomes was identified.Western blotting was used to detect the expression of exosome markers Alix,TSG101 and CD63.4.Effects of plasma exosomes on SW480:(1)The isolated plasma exosomes were labeled with fluorescent dye Di I,and then co-cultured with SW480 cells.The uptake of exosomes was observed under a confocal microscope.(2)CCK-8 assay was performed to detect the effect of plasma exosomes on the proliferation of SW480 cells.(3)Transwell assay was used to detect the effect of plasma exosomes on the migration and invasion of SW480 cells.(4)Western blotting analysis was conducted to detect the influence of exosomes on related signaling pathways.(5)The role of Akt /m TOR signaling pathway in the induction of colon cancer cell proliferation and migration by exosomes was verified by m TOR inhibitors.Result:1.Isolation and identification of cell-derived exosomes:(1)NTA and TEM results showed that the exosomes of cell culture supernatant isolated by ultracentrifugation were small vesicles with a diameter of about 120 nm,which were small vesicles with membrane structure.(2)Western blotting detected the expression of exosome markers Alix,TSG101 and CD63,suggested that exosomes from cell lines have been successfully isolated by ultracentrifugation.2.Effects of exosomes from different cell lines on SW480:(1)Exosomes derived from NCM460 and SW620 cells were taken up by SW480 cells.(2)Cell-derived exosomes promoted the migration and invasion of SW480 cells in different degrees,of which exosomes from colon cancer cells had a stronger promoting effect than from normal intestinal epithelial cells.(3)Exosomes could affect the Akt/m TOR signaling pathway and EMT procession in recipient cells3.Evaluation of methods for plasma-derived exosomes:(1)NTA results showed that the exosomes separated by precipitation method were larger in size(about 170 nm)than those isolated by ultracentrifugation(about 100 nm).The exosomes isolated by three SEC methods(Sepharose CL-2B,Sepharose CL-4B,q EV)were concentrated in 9 to 11 fractions.There was no significant difference in particle size,which was about 110 nm.(2)The results of BCA assay showed that the recovery of the q EV column was about 58.7%,Sepharose CL-2B was about 53.7%,while Sepharose CL-4B was about 50.5%,among which the recovery rate of ultracentrifugation was the lowest.(3)TEM results showed that the exosomes obtained by ultracentrifugation and SEC method were of high purity,and there was no significant difference among the three kinds of SEC filter columns.Exosome purity obtained by ultracentrifugation method was lower,while exosome obtained by precipitation method was the lowest.(4)The expression level of plasma exosome markers Alix,TSG101 and CD63 could be detected by western blotting in all the three isolation methods.4.Effects of plasma-derived exosomes on SW480:(1)Plasma-derived exosomes were taken up by SW480 cells.(2)Plasma-derived exosomes promoted the proliferation,migration and invasion of SW480 cells.What’s more,the plasma-derived exosomes from colon cancer patients have a stronger promoting effect than the healthy controls.(3)Plasma-derived exosomes can activate the Akt /m TOR signaling pathway in colon cancer cells.Furthermore,the addition of m TOR inhibitors could inhibit the proliferation and migration ability of SW480 cells induced by plasma-derived exosomes.Conclusion:1.Exosomes derived from colon cancer cells can promote the migration and invasion ability of SW480 cells,which may play a role through the Akt /m TOR signaling pathway and EMT procession.2.Plasma exosomes obtained by SEC are with high recovery and purity,which is an effective method for isolating plasma exosomes.3.Exosomes derived from plasma of colon cancer patients cann promote the proliferation,migration and invasion ability of SW480 cells and may associated with the Akt/m TOR signaling pathway. |
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