Expression And Function Of Galectin-3in Macrophages Induced By C. Albicans

Part Ⅰ:Candida albicans on the activation of macrophage U937Purpose:Candida albicans on the activation of the human macrophage cell line U937cells were observed.Methods:observe the concentration of NO and Galectin-3expression and cell viability in cell culture mediumd after Candida albicans stimulate human macrophage cell line U937cells at different time points and different concentrations.Results:1. Candida albicans a final concentration of expression and cell survival factor in macrophage cell line U937:107PFU/mL and below promote cell growth, the relative survival rate increased, NO and Galectin-3expression increased in the cells, in each groupthere was statistically significant (P<0.05);108PFU/mL and above, inhibits cell growth, the relative survival rate decreased,NO and expression of Galectin-3decreased in cells, among the three groups was statistically significant(P<0.05).2. Candida albicans'duration of action and the cell survival and factor expression in macrophage cell line U937:Candida albicans for3h and above, can promote cell growth, NO and Galectin-3expression in cells enhanced statistics among the three groupssignificance (P<0.05); Candida albicans for3h and the following little impact on cell growth, cell NO and expression of Galectin-3did not change significantly, no significant difference between the groups (P>0.05).Conclusion:1. Candida albicans concentration of107PFU/mL and below stimulate human macrophage cell line U937cell activation,was positively correlated with the concentration of NO and Galectin-3expression and cell viability.2.Candida albicans in the role of time3h and above activation of the human macrophage cell line U937cells. Part Ⅱ The expression and function of GaIectin-3in Candida albicans-induced human macrophage cell line U937cells.Objective:To observe the expression of Galectin-3in Candida albicans-induced human macrophage cell line U937cells and to explore its function.Methods:Build galectin-3gene knockdown U937macrophage cell line model Expression of Galectin-3-shRNA silencing by lentiviral vector-mediated,vitro evaluation and observed the expression of galectin-3in human macrophage cell lineU937cells to Candida albicans infection, cell viability and cell concentration of NO, IL-8, TNF-a, of COX2, TLR2and TLR4in the culture medium expression changes;the determination of the combination of Galectin-3and Candida albicans;the laser scanning confocal microscope Galectin-3and of TLR2and TLR4expression and distribution.Results:1.using the universal primer PCR detection of the colony. Obtained four positive clones, sent on the3rd samples were verified by sequencing and the results show that for galectin-3shRNA sequence has been successfully connected to the lentiviral vector.2. Quantitative PCR and Western blot analysis showed that Galectin-3expression within the cell by a significant knockdown. 3. Promote cell growth of Candida albicans to stimulate U937cells, IL-8, TNF-a, COX2, TLR2and TLR4increased expression, the level of NO upward, statistical significance between the groups. The Silence expression of Galectin-3cell growth was inhibited, the relative survival of cells decreased, IL-8, TNF-a, COX2, TLR2and TLR4expression increased, the level of NO upward,statistical significance between the groups.4. Binding assay results increased with the increase in the concentration of Candida albicans, Galectin-3and the degree of integration of the Candida albicans corresponding to deepen, when the concentration of107PFU/mL and above significantly increased, statistical significance between the groups.when the concentration of109PFU/mL, compared with108PFU/mL of group, no statistical significance. Silence expression of Galectin-3got similar results. However, the degree of integration than the control group decreased significantly.Conclusion:1. This experiment was successfully constructed lentiviral vector-mediated Expression of Galectin-3-shRNA silencing gene human U937macrophage cell line model.2.Candida albicans stimulate human macrophage cell line U937cells, Galectin-3has the negative regulation of NO, IL-8, TNF-α, COX2, TLR2and TLR4expression in cell culture medium concentration of NO, IL-8, the regulation of TNF-alpha, of COX2, TLR2and TLR4expression after Candida albicans stimulate human macrophage cell line U937cells,was negatively correlated with cell viabilitywere positively correlated.3.Galectin-3can directly binds to Candida albicans. Part Ⅲ Mechanism analysis of galectin-3in C.albicans-induced human macrophage cell line U937cellsObjective:To investigate the mechanism of galectin-3in Candida albicans-induced human macrophage cell line U937cells. Methods:Western blot (Western blot) to detect cell FAS, of NF-κB, JNK, and ERK1/2protein content.Results:1. In the case of Galectin-3was inhibited Candida albicans infection will result in the upregulation of FAS and NF-κB protein in the cells.2. In the case of Galectin-3was inhibited Candida albicans infection can cause cell expression of phosphorylated JNK upregulation and phosphorylation of ERK1/2protein downregulation.Conclusion:1. The galectin-3was involved in the response to activation of the MAPK pathway in Candida albicans induced human macrophage cell line U937cells.2. Galectin-3was involved in the anti-apoptotic role by regulating the expression of Fas and NF-κB3. Galectin-3is a key molecule for inhibition of Candida albicans infection and potential therapeutic targets of invasive Candidiasis.