| Summary: | 碩士 === 國立陽明大學 === 藥理學研究所 === 95 === Many complicated mechanisms affect the activation of microglia and macrophage after the spinal cord injury and influence the outcome of recovery. Our preliminary data showed large numbers of activated macrophages were recruited in spinal cords after a repair strategy of peripheral nerve grafts with acidic fibroblast growth factor (aFGF) treatment. In addition, the expression of arginase I (Arg I) and polyamines were significantly upregulated in repaired spinal cords. These findings indicated that some molecular mechanisms triggered by the full repair procedure in the early stage of spinal cord injury may be important to functional recovery.In this study, we investigated the expression patterns of Arg I and polyamines in in vitro macrophage culture and in vivo animal models.
Western blotting analysis showed that IL-4 (10 ng/ml) can stimulate macrophage cell line, RAW 264.7, to arginase I-positive alternatively activated macrophages but not LPS (1 mg/ml) or IL-1b + TNFa + INFg. IL-4、LPS and IL-1b + TNFa + INFg along or combined with aFGF (50 ng/ml) induced different level of phosphorylated proteins expression in signaling pathways of activated macrophage. aFGF alone can't significantly induce arginase I and iNOS expression in macrophages and there were no synergistic effects on the expression of arginase I and iNOS when IL-4、LPS or IL-1b + TNFa + INFg combined with aFGF treatment. Q-PCR results showed that, 6 hours after stimulation, combination of aFGF, IL-4 induced spermidine synthase mRNA expression. 6 to 48 hours after stimulation, with/without combination of aFGF, IL-4 induced arginase I mRNA expression, compared with the same time groups. LPS or IL-1b + TNFa + IFNg induce arginase II or ornithine decarboxylase mRNA expression in 24 or 12 hours. 6 to 48 hours after stimulation, with/without combination of aFGF, IL-1b + TNFa + IFNg induced iNOS mRNA expression, compared with the same time groups. HPLC analysis results show that polyamines released by combination of aFGF, IL-4 induced macrophages were higher than LPS induced macrophages after 24 and 48 hr stimulation. Addition of arginase I and ornithine carboxylase inhibitors (such as nor-NOHA and DFMO) in repaired spinal cords reduced the recovery of animal behavior after 8 weeks long-term observation. These data revealed that the expression of polyamines would be an essential key element for our repair strategy. Alternatively activated macrophages were suggested to be important to spinal cord repair and neural regeneration.
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