Study On The Endocytosis Mechanism Of S100B In Bone Marrow Mesenchymal Stem Cells

Chapter? Time-dependent internalization of S100B by mesenchymal stem cells and its intracellular transportation processObjective: To investigate whether exogenous S100B can be internalized into bone marrow mesenchymal stem cells and its intracellular transportation process.Methods: The expression of specific surface markers(CD29,CD90,CD71,CD45,CD34)of BMSCs at passage 4 were identified with flow cytometric assays.SDS-PAGE electrophoresis and Western blot were used to identify the S100B-Alexa488(protein-dye)conjugate to verify whether the S100B recombinant protein was successfully labeled.Dextran is a fluid-phase endocytic marker,BMSCs were incubated with S100B-Alexa488 and fluorescently labeled dextran(Dextran-TMR)at the same time,by using confocal microscope,we monitored the time-dependent internalization of S100B-Alexa488 and Dextran-TMR in detail,we evaluated the number,mean fluorescence intensity(MFI)and colocalization level of fluorescent vesicles internalized per cell at 4sequential time points.To explore whether the intracellular transportation of S100B-Alexa488 passed the endosomal pathway,the co-localization level of S100B-Alexa488 with early endosomal marker or late endosomal marker was detected by indirect immunofluorescence assays with the help of confocal microscopy.After being incubated with S100B-Alexa488 for different periods of time,BMSCs were stained with Lyso-Tracker Red,a lysosomal red fluorescent probe,and the colocalization level of intracellular S100B-Alexa488 and lysosomes was detected by confocal microscope to study whether the intracellular transportation of S100B-Alexa488 passed the lysosomal pathway.Results: Flow cytometry results showed that the expression of specific surface markers(CD29,CD90,CD71,CD45,CD34)of BMSCs at passage 4 were 95.2%,98.3%,98.3%,1.56%,1.05%,respectively,indicating that the purity of BMSCs was relatively high and can be used for following research.The results of SDS-PAGE electrophoresis showed that the Alexa488 dye successfully labeled the S100B protein and there was almost no free dye in the conjugate.Western blot showed that the S100B protein in the conjugate could be recognized by the S100B primary antibody,indicating the S100B protein retained its antigen integrity.The S100B-Alexa488/Dextran-TMR co-staining results showed that the number and MFI of the two fluorescent vesicles increased roughly with time.S100B can be taken up by MSCs in a time-dependent manner like Dextran-TMR.Indirect immunofluorescence assays showed that the colocalization index at 12 h of S100B-Alexa48 and LAMP1 was significantly higher than that at 24 h,however,the colocalization index of S100B-Alexa48 and EEA1 at 12 h was not significantly different from that of 24 h.The internalized S100B-Alexa488 may pass through early endosomes and gradually transfer to late endosomes.The S100B-Alexa488/lysosomal co-staining experiments results showed that internalized S100B-Alexa488 accumulated in lysosome-like structures,and the colocalization index of S100B-Alexa488 and lysosomal was above 0.6within 24 h,indicating there was a high colocalization between the two fluorescent probes,the number of S100B-Alexa488-positive vesicles co-localized with lysosomes increased over time,and S100B-Alexa488 was transported intracellularly through the lysosome pathway.Conclusion: Exogenous S100B is internalized into bone marrow mesenchymal stem cells in a time-dependent manner and transported intracellularly through endosomal and lysosome pathways.Chapter? The endocytosis mechanism of S100B in BMSCsObjective: To study the effect of clathrin and dynamin on the endocytosis of S100B-Alexa488 and explore whether the internalization of S100B-Alexa488 depends on the clathrin-mediated endocytosis,which provides the possible interventions and scientific basis to avoid the malignant transformation of BMSCs in the treatment of glioma.Methods: We established a CCK8 standard curve to determine the optimal number of cells and the reaction time of CCK8 working solution.The relative survival rate of cells treated with clathrin inhibitor Pitstop-2and dynamin inhibitor Dyngo-4a was detected using CCK8 methods to determine the optimal concentration of drugs.BMSCs were incubated with S100B-Alexa488 and fluorescent-labelled transferrin(Transferrin-Dylight649)simultaneously whether in DMSO(0.2%)treated cells or untreated cells.Confocal microscope was used to detect the number and MFI of the two fluorescent probes-positive vesicles to study whether DMSO has an adverse effect on the endocytosis of the two fluorescent probes.BMSCs were pretreated with Pitstop-2,an inhibitor for clathrin,Dyngo-4a,an inhibitor for dynamin,and small interfering RNA targeting CHC or DNM2,respectively,then cells were incubated with S100B-Alexa488 and Transferrin-Dylight649 at the same time.Then the number and MFI of the two fluorescent vesicles were detected by confocal microscope and analyzed quantitatively in the treated groups and the control group.Transferrin was used as a parallel control to investigate the effect of clathrin and dynamin on the internalization of S100B-Alexa488.Results: According to the CCK8 standard curve,the number of cells should be 10000 / well when detected,and the reaction time of CCK8 working solution was about 4 h.According to the relative survival rate of50% of cells,the determined concentrations of Pitstop-2 and Dyngo-4a are5?M and 20?M,respectively,which have little effect on cell status.After treatment with the clathrin inhibitor Pitstop-2 or the dynamin inhibitor Dyngo-4a,the number of Transferrin-Dylight649-positive vesicles decreased significantly compared with the control group,indicating the two inhibitors inhibited clathrin and dynamin,respectively.The number of S100B-Alexa488-positive vesicles was significantly reduced than that of control cells,indicating that the internalization of S100B-Alexa488 mainly depends on clathrin dynamin.After treatment with small interfering RNAs targeting CHC or DNM2,the number of Transferrin-Dylight649-positive vesicles decreased significantly compared with the control group,indicating the two small interfering RNAs inhibited clathrin and dynamin,respectively,moreover,the number of Alexa488-S100B-positive vesicles was significantly reduced compared with the control group,further indicating that the endocytosis of S100B-Alexa488 mainly depends on clathrin and kinesin.Conclusion: The endocytosis of S100B-Alexa488 in BMSCs depends on clathrin and kinesin.It mainly depends on clathrin-mediated endocytosis.