A New Method For Extracting And Isolating Endothelial Progenitor Cells And The Effect Of SIRT6 On B Cell Development And BCR Signal

PART ?A NEW METHOD FOR EXTRACTING AND ISOLATING ENDOTHELIAL PROGENITOR CELLSObjective: To explore a new method for the extraction and isolation of endothelial progenitor cells(EPCs),and use the method to increase the number of EPCs cells and proliferative activity.Methods: EPCs were isolated from fetal rat lung and verified with morphology,cell surface markers,phagocytosis and blood vessels formation.The proliferation activity of EPCs which were extracted from bone marrow were compared.Result: The morphological characteristics of the cells isolated from fetal rat lung were consistent with those of EPCs.The cells can express the specific markers of EPCs,such as CD31,CD34,CD133,VEGFR2 and so on.Also,these cells could engulf Dil-ac-LDL and FITC-UEA-1 and form lumen structure in vitro.All in a word,these cells extracted from fetal rat lung were authenticated to be EPCs.The proliferative activity of EPCs extracted from fetal rat lung was significantly higher than that from bone marrow.Conclusion: It's feasible to extract EPCs from fetal rat lung,and to provide a reliable cell source for further experiments.PART ?THE EFFECT OF SIRT6 ON B CELL DEVELOPMENT AND BCR SIGNALObjective: To explore the effect of SIRT6 on B cell development and BCR signal,and to clarify its mechanism.Methods: The SIRT6 f/f CD2 iCre mice(conditional gene knockout mice)model was constructed,and the SIRT6 RNA level and SIRT6 protein expression were detected by Real-time PCR and Western blotting.Using wild-type(WT)mice and SIRT6 f/f CD2 iCre mice as models,bone marrow cells and spleen lymphocytes were extracted and stained by flow cytometry to observe the difference of B cell subsets between the two mice,in order to observe the effect of SIRT6 on B cell development.Using WT mice and SIRT 6f/f CD2 iCre mice as models,spleen B cells were extracted.BCR-(Fab)2-SA was combined with water bath activation at 37 C to simulate the activation process of BCR in vivo.Protein was extracted from activated B cells and Western blotting was performed to observe the changes of BCR signal-related molecules.Result: Compared with WT mice,SIRT6 f/f CD2 iCre mice showed lower SIRT6 RNA expression in B and T cells,lower SIRT6 protein expression in B cells,and no significant decrease in SIRT6 protein expression in T cells.In SIRT6f/fCD2 iCre mice,the proportion of pre-B and immature B cells increased while that of circulating mature B cells decreased.In SIRT-6f/fCD2 iCre mice,the proportion of MZ-B cells to total B cells decreased,while that of GC-B cells to total B cells increased,while that of Fo-B cells did not change significantly.In SIRT6f/f CD2 iCre mice,the p CD19 signal was enhanced and the degradation was delayed,while the p SHIP signal was decreased.Conclusion: The lack of SIRT6 results in the stagnation of B cell development in bone marrow at the late pre-B and immature B cell stages,which leads to the decrease of circulating mature B cells.The absence of SIRT6 may also lead to a decrease production of low affinity antibodies,but enhances memory humoral immune response and long-term antibody production,and the absence of SIRT6 enhances B cell activation.