Summary: | 碩士 === 國立臺灣大學 === 園藝學研究所 === 93 === Taiwanofungus camphoratus, a traditional and potential medicinal mushroom in Taiwan, is known to have immunomodulatory and antitumor activities. ACA1, which is a glycoprotein obtained from T. camphoratus and is capable to activate mouse macrophages and splenocytes, has been cloned into pET32 vector and successfully expressed in E. coli to obtain normal protein, rACA1. Here, we investigate the bioactivity of rACA1 on murine macrophages and the membrane receptor and the intracellular signaling pathway responsible for the activation of macrophages by rACA1. rACA1 is demonstrated to the production of nitric oxide (NO) and TNF-alpha both in RAW 264.7 cells and peritoneal macrophages from normal mice. To investigate the membrane receptor involved in the activation of TNF-alpha production, we further examine the effects of rACA1 on the production of TNF-alpha in mouse peritoneal macrophages isolated from wild type C57BL/6 and from functional Toll-like receptor 4 (TLR4)-deficient C57BL/10ScN mice. The results show that rACA1 induces TNF-alpha production by macrophages isolated from both C57BL/6 and C57BL/10ScN mice. However, anti-TLR2 mAb is demonstrated to block the activation of rACA1 toward peritoneal cells, which indicates that TLR2 is highly involved in rACA1 signaling. Further we examine the effect of rACA1 on the activation of an adapter protein, MyD88, which is a downstream mediator of TLR2. rACA1 of macrophages activation was obstructed by dominant-negative MyD88 to produce TNF-alpha. Taken together, these results suggest that rACA1-mediated induction of TNF-alpha production in macrophages is mediated, at least in part, by TLR2/MyD88 signaling pathway.
|