| 摘要: | 巨噬細胞在宿主抵抗發炎及細菌感染的過程中不但扮演了一個重要的角色，更直接參與了細菌、細胞碎片的吞噬、抗原的處理與表達、及細胞激素、活性氮化物的分泌與釋出。內毒素是葛蘭氏陰性菌細胞壁的成份之一，目前已知其可以造成動物體內諸如局部發炎、抗體產生及敗血症等生理與病理反應。在宿主抵抗細菌感染的過程中，巨噬細胞常常也是內毒素的作用標的。已有實驗顯示in vitro 下為內毒素所作用的巨噬細胞與受細菌感染動物體內之巨噬細胞有諸多類似之生理、生化反應。因此，前者可做為研究動物受細菌感染後體內巨噬細胞如何因應的模型。 c-Src 是一個分子量為60 kDa 的nonreceptor tyrosine kinase，其家族成員包括了Yes, Fyn, Lck, Lyn, Fgr 及Hck。由於內毒素的活化巨噬細胞會為tyrosine kinases inhibitors (i.e. herbmycin) 所抑制，加上Lyn、Fgr 及Hck 三個表現於巨噬細胞的Src 家族成員 (myeloid-specific Src members) 會因LPS 的刺激而活化，故這三者被認為是LPS 活化巨噬細胞的responsible kinases。由於Lyn、Fgr 及Hck 三者皆剔除的實驗組老鼠(triple knockout mice) 與正常老鼠的巨噬細胞在內毒素刺激後顯現程度相當的活化，故科學界認為在內毒素活化巨噬細胞的過程中，理應有nonmyeloid-specific Src member(s) 或其他tyrosine kinase(s) 的參與。的確，我們已證明無論in vitro 或in vivo，內毒素皆可使巨噬細胞的c-Src 表現量增加；且內毒素的活化巨噬細胞會因PP2 (Src kinases 的抑制劑) 的存在而受到抑制。因此我們認為c-Src 在內毒素所引發巨噬細胞的活化過程中扮演一舉足輕重的角色(Molecular Immunology, 2006; in press)。日前在深入探討內毒素誘增c-Src 的機轉時，我們發現 LPS-induced protein tyrosyl phosphorylation 及細胞位移會因iNOS 抑制劑(i.e. L-NAME) 的存在而下降；且c-Src 的表現量會因NO donor (i.e. SNAP) 的刺激Raw264.7 細胞而增加。因此，針對這些發現我們擬定下列幾個主題進行研究。 (1) 確定巨噬細胞的c-Src 表現量無論in vitro 或in vivo 皆會因一氧化氮的刺激而增加。 (2) 確定巨噬細胞的c-Src 表現量無論in vitro 或in vivo 皆會因 cGMP 的刺激而增加。 (3) 在巨噬細胞內，那些訊息傳遞路徑參與了一氧化氮對c-Src 的誘增？ (4) 探討在巨噬細胞內，一氧化氮誘增c-Src 的生理意義。
As important effectors of innate immunity, macrophages play a crucial role in the host response to inflammation and bacterial infection. During these processes, macrophages are mainly participated in the phagocytosis of bacteria and host cell debris, processing and participation of antigens, and the secretion and release of reactive nitrogen intermediates as well as inflammatory cytokines. Lipopolysaccharide (LPS), a membrane component of Gram–negative bacteria, induces a variety of physiological and pathological responses such as local inflammation, antibody production and septic shock. During bacterial infection, macrophages are major cellular targets for LPS action. In vitro exposure of macrophages to LPS mimics many of the effects bacteria have on macrophages in vivo. Thus, the former becomes a model to study the responsiveness of macrophages in bacteria-infected animals. c-Src is a 60 kDa nonrecptor tyrosine kinase whose family members include Yes, Fyn, Lck, Lyn, Fgr and Hck. Among them, Lyn, Fgr and Hck that specifically express in macrophages are thought to be essential in LPS-induced macrophage activation. However, like those from normal mice, macrophages derived from triple knockout mice (mice that with three-combined deficiency of Hck, Fgr and Lyn) could still respond to LPS. Interestingly, we observe the upregulation of c-Src in Raw264.7 and peritoneal macrophages (PEMs) by LPS. And this LPS-mediated c-Src induction is also observed in macrophages recovered from LPS-challenged rats. Notably, PP2 attenuates the ability of PEMs to elicit COX-2 expression and nitric oxide production in response to LPS. Thus, c-Src, with its LPS induction, should have an unperceived role in transmitting LPS signaling (Molecular Immunology, 2006, In press). In an attempt to elucidate the mechanisms responsible for the enhancement of c-Src upon exposure to LPS, we observe that L-NAME (an iNOS inhibitor) can abrogate LPS-induced protein tyrosyl phosphorylation as well as cell migration. Remarkably, the expression of c-Src can be upregulated in response to NO donors (i.e. SNAP) in Raw264.7 macrophages. Based on these findings, the following specific aims will be addressed and pursued. (1) Investigation of the influence of nitric oxide on the expression of c-Src in macrophages both in vitro and in vivo. (2)Study the influence of nitric oxide on the expression of c-Src in macrophages both in vitro and in vivo. (3)Dissect the molecular mechanisms underlying the enhancement of c-Src mediated by nitric oxide. (4) Investigation of the physiological significance of LPS-induced c-Src expression in macrophages. |
