以條件式基因剔除小鼠探討Cullin 4B缺失對於先天性免疫反應之影響

Study on the influence of Cullin 4B defect on the innate response of the conditional knock-out mice

洪明宏(Ming-Hung Hung)

指導教授 ： 莊雅惠

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.02738

摘要

Cullin 4B (Cul4B)屬於Cullin 4B-RING E3 ubiquitin ligase (CRL4B) complex中的骨架蛋白，可以將細胞內蛋白質泛素化(ubiquitination)使其被proteasome降解或執行其他功能。Cul4B調控的蛋白質包括調節細胞週期與DNA複製相關的蛋白質。人類的Cul4B基因突變時會罹患X-linked intellectual disability (XLID)，這類病人的周邊血液中單核球增加，表示Cul4B突變可能導致單核球增生。有研究發現以shRNA調降巨噬細胞株(RAW264.7)中Cul4B的表現後，在LPS刺激下巨噬細胞株產生的TNF-α減少。綜合以上結果表示Cul4B可能會影響單核球或巨噬細胞的發育及免疫功能。然而，單核球與巨噬細胞對於先天性免疫十分的重要，在此我們製造出骨髓性特異性Cul4B缺失(LysMCreKI/KI Cul4bf/y or f/f; CKO)小鼠來研究Cul4B缺失對於先天性免疫的影響。首先，我們確認CKO小鼠的Cul4B的確特異性缺失於骨髓性白血球中，與正常小鼠比較，CKO小鼠的骨髓細胞數與周邊血全血球計數上也無明顯的差異，表示CKO小鼠沒有明顯血球發育問題。然而我們將LPS注射入小鼠腹腔引起急性腹膜炎後，CKO小鼠體重下降幅度較正常小鼠大，並且在腹腔當中有較多的免疫細胞。但單一免疫細胞分泌發炎細胞激素(TNF-α, IL-6)的能力較差。而在體外細胞實驗中證實缺少Cul4B的巨噬細胞經LPS刺激後所分泌TNF-α及IL-6較少，但CKO的巨噬細胞比起正常小鼠較易進行DNA複製而加快細胞週期而使增生速率加快。因此，骨髓特異性Cul4B缺失小鼠在LPS誘發腹膜炎的反應會比較嚴重。而在細胞實驗中發現Cul4B的缺失影響巨噬細胞DNA複製進而使其加速增生與抑制其分泌發炎細胞激素。我們的研究成果發現Cul4B或可能其他Cullin家族的成員在免疫反應中亦扮演角色。

關鍵字

先天性免疫反應 ； 骨髓特異性缺失小鼠 ； 腹膜炎小鼠模式 ； Cul4B ； 巨噬細胞

並列摘要

Cullin 4B (Cul4B), one member of cullin family, is a scaffold protein of Cullin4B-RING-E3 ligase complex which ubiquitinate intracellular protein. The targets for cul4B include cell cycle regulation proteins and some DNA replication-related molecules. Patients with defect or mutation of Cul4B have increased frequency and numbers of peripheral monocytes. In addition, RAW264.7 macrophages with knockdown of cul4B expression by shRNA secrete lower level of TNF-α. These results indicated that Cul4B would affect the development and immune function of monocytes/macrophages which are very important for innate immunity. In this study, we generated a myeloid-specific Cul4B deficient mice (LysMCreKI/KI Cul4bf/y or f/f; CKO) to investigate the influence of Cul4B defect on innate immunity. At first, we confirmed that Cul4B gene was deleted in only myeloid cells of CKO mice. We found the number of bone marrow cells and complete blood count and differential count of peripheral blood were no difference in CKO and control mice, suggesting that Cul4B defect on myeloid cells did not affect the development of blood cells. However, after an intraperitoneal injection of LPS, significantly decreased in the body weight and increased immune cells in peritoneal cavity of CKO mice were observed. Surprisingly, each peritoneal exudate cell in LPS injected CKO mice produced fewer proinflammatory cytokines TNF-α and IL-6 than control mice. Furthermore, bone marrow derived macrophages from CKO mice secreted fewer TNF-α and IL-6 upon LPS stimulation but proliferated faster due to the cell cycle acceleration. Given our findings, myeloid-specific Cul4b deficiency worsened LPS induced acute peritonitis. In macrophages, Cul4B deficiency enhanced DNA replication and proliferation but decreased production of proinflammatory cytokines. Our data highlights the new role of Cul4B and maybe other members of cullin family in the immune responses.

並列關鍵字

innate immune response ； LysMCre ； myeloid-specific knockout mice ； experimental peritonitis mouse model ； Cul4B ； macrophage

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