2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646583摘要：Cul4B 是泛素(ubiquitin)系統的一員，泛素依賴性蛋白分解為細胞調控蛋白活性基本的機制。最近臨床發現Cul4B基因點突變的病人會造成遺傳性心智遲緩，此種病人其血液單核球數目及比例上升，代表CUL4B基因在造血作用中扮演重要的角色。因Cul4B為性聯遺傳基因且小鼠胚胎幹細胞來自公鼠胚胎，因此Cul4B基因剔除胚胎幹細胞無法存活。在此我們利用Cul4B條件式基因剔除小鼠來探討Cul4B在骨髓性白血球的發育及功能之角色，以及巨噬細胞及嗜中性球Cul4B基因缺失下T 細胞及B 細胞的功能。子目標如下：1. 建立巨噬細胞及嗜中性球Cul4B基因剔除小鼠（Cul4Bf/yLysMcreKI/KI mice）2. 研究Cul4B在單核球及嗜中性球發育的角色a. 分析Cul4Bf/yLysMcreKI/KI小鼠骨髓之單核球及嗜中性球的細胞數目、成熟及細胞凋零情況b. 分析周邊血嗜中性球及單核球的細胞數目3. 研究Cul4B在嗜中性球功能上的角色a. 評估不同TLR刺激劑對活化Cul4Bf/yLysMcreKI/KI小鼠嗜中性球的能力b. 評估Cul4Bf/yLysMcreKI/KI mice嗜中性球吞噬作用的能力4. 研究Cul4B在巨噬細胞功能上的角色a. 分析Cul4Bf/yLysMcreKI/KI mice對LPS注射產生之免疫反應b. 分析Cul4B基因缺失下的巨噬細胞其表現型及分泌發炎分子與吞噬作用之能力5. 研究Cul4Bf/yLysMcreKI/ KI mice的B細胞免疫反應a. 分析周邊血、脾臟及肝的B細胞數目b. 建立B細胞剔除小鼠來研究Cul4B剔除對B細胞發育及功能的直接影響6. 研究Cul4Bf/yLysMcreKI/KI mice的T細胞免疫反應a. 分析周邊血、脾臟及肝的T細胞數目b. 建立T細胞剔除小鼠來研究Cul4B剔除對T細胞發育及功能的直接影響<br> Abstract: CUL4B is a component of the ubiquitin system. Ubiquitin-dependent proteolysis is a fundamental cellular mechanism for regulating protein activity. Point mutations in CUL4B were identified recently in patients with syndromic X-linked mental retardation (XLMR). Increased frequency and numbers of monocytes in the peripheral blood were observed in patients with XLMR, indicating that CUL4B plays a critical role in hematopoiesis. We have succeeded in generating conditional Cul4B-floxed mice. Because Cul4B is an X-linked gene and the mouse embryonic stem (ES) cells are derived from male embryos, we found that KO ES cells are not viable, and therefore, conditional KO is the appropriate strategy. With the conditional Cul4B-floxed mice, we will investigate the role of Cul4B in myeloid development and function as well as the adaptive immunity in mice devoid of Cul4B in monocyte/macrophage and neutrophils. The specific aims are as followed:1.Generation of mice in which Cul4B is deficient in monocyte/macrophage and neutrophils (Cul4Bf/yLysMcreKI/KI mice).2.To study the role of Cul4B in monocyte/macrophage and neutrophil development.a.Analyze the cell numbers, maturation status, and apoptosis of neutrophils and monocytes in bone marrow of Cul4Bf/yLysMcreKI/KI mice.b.Analyze the cell numbers of neutrophils and monocytes in peripheral blood.3.To study the role of Cul4B in neutrophil function.a.Assess the capacity of various TLR agonists to activate neutrophils in Cul4Bf/yLysMcreKI/KI mice.b.Assess the capacity of phagocytosis of neutrophils from Cul4Bf/yLysMcreKI/KI mice.4.To study the role of Cul4B in monocyte/macrophage function.a.Analyze the effect of Cul4B deficiency on LPS responsiveness in vivo.b.Analyze the activation status of peritoneal macrophages in Cul4Bf/yLysMcreKI/KI mice on the basis of surface phenotype and production of inflammatory mediators.c.Assess the capacity of phagocytosis of macrophages from Cul4Bf/yLysMcreKI/KI mice.5.To study the B cell immune responses in Cul4Bf/yLysMcreKI/KI mice.a.Analyze the cell numbers of B cells in peripheral blood, spleen, and liver to confirm the increased number of B cell in the periphery.b.Generate B cell-specific Cul4B deficiency mice to study the direct effect of Cul4B in B cell development and function6.To study the T cell immune responses in Cul4Bf/yLysMcreKI/KI mice.a.Analyze the cell numbers of T cells in peripheral blood, spleen, and liver.b.Generate T cell-specific Cul4B deficiency mice to study the direct effect of Cul4B in T cell development and function.