| During the last decade characteristics and functions of B-1 cells have attracted a lot attention. B cells is important immune system components, and current studies suggest that B cells could be divided into at least two subsets such as B1 and B2 cell, B1 cells in the abdominal cavity which has the characteristics of natural immune cells, expressing phagocytosis-related surface molecules Mac-1, F4/80 and so on, playing an important role in the natural immune response against infection. Early our experimental group had found that peritoneal B1 cells could swallow staphylococcus aureus, and had a clear role of bacterial and fungal infections. Bactericidal mechanisms of professional phagocytes is divided into two types,one which pathogens are killed by lysosomal enzymes, the other which is considered as a bactericidal mechanism of oxidation, and pathogens are killed by oxygen free radicals such as NO which is produced through the respiratory burst. Whether there is oxide sterilization mechanism in phagocytosis and sterilization process of mouse peritoneal B1 cells has not been clearly established. In order to further explore the funtion of NO in the sterilization process of B1 cells, its mechanism is given a preliminary study in this experiment.AIM:This study was designed to culture B1 cells from mice peritoneal with Staphylococcus aureus(SA) in vitro, detect nitric oxide(NO)synthesis and the expression of inducible nitric oxide synthase(iNOS)and cytokine mRNA. Observe the survival rate of intracellular SA after inhibition of iNOS function with aminoguanidine(AG)in vitro, and analyze the role of NO in the sterilization mechanism of peritoneal B1 cells.METHODS:(1) Peritoneal cells and spleen cells obtained from normal C57BL/6 mice was isolated and purified with PE/Cy5-rat anti mouse CD19 magnetic beads. Finnally obtain peritoneal CD19+cells (B1 cells) and peritoneal CD19-cells (macrophages). Detect the efficiency of magnetic beads-on separation by flow cytometry and these cells selected with magnetic beads is co-cultured with SA, receive cells at different times and detect the changes of NO synthesis; (2) Select B1 cells co-cultured with Staphylococcus aureus at different times, extract total RNA conventionally, By Real Time-PCR, analyze the expression of iNOS and cytokines in each group; (3) Co-cultured B1 cells with Staphylococcus aureus, meanwhile inhibiting iNOS with AG medium, observe the survival rate of intracellar SA.RESULTS:(1) By Flow cytometry, we found the efficiency of sorting B1 cells by magnetic bead was higher. After Co-culturing B1 cells with Staphylococcus aureus, by the nitrate reductase assay, we found that the amount of NO production was significantly increased compared with the control group and was proportional to incubation time; (2) By Real Time-PCR, after stimulation of Staphylococcus aureus, it was found at different time that the expression of iNOSmRNA in B cells significantly increased, IL-4, IL-10 expression was significantly reduced compared with the control group, TNF-α, IFN-γexpression was significantly higher compared with the control group; (3) The experimental results show that the survival rate of intracellar Staphylococcus aureus was significantly higher than that of aminoguanidine group in which iNOS was inhibited by AG in vitro.CONCLUSION: Stimulated by Staphylococcus aureus, peritoneal B1 cells significantly increased NO synthesis and iNOS expression, meanwhile IL-4,IL-10,TNF-α,IFN-γand other cytokines mRNA expression levels of B1 cells have a significant change. After iNOS had suppressed by aminoguanidine, the survival rate of intracellar Staphylococcus aureus was significantly increased. So we can get a conclusion that B cells received foreign antigen stimulation in mice increased the synthesis of NO, and iNOS specific inhibitor can inhibit its bactericidal function, It inferred that there be NO-dependent, oxidative bactericidal mechanism in mouse B1 cells. |
PDF Full Text Request