鹼性磷酸酶在斑馬魚的巨噬細胞分化過程中扮演的角色

在斑馬魚中有組織非專一性鹼性磷酸酶跟腸道鹼性磷酸酶兩種，在過去的文獻中已經得知組織非專一性鹼性磷酸酶(TNAP)主要參與了斑馬魚早期胚胎發育，TNAP會大量地表現於斑馬魚的腎臟，腎臟同時也是斑馬魚的主要造血器官，從先前的研究得知，TNAP 的缺乏會影響斑馬魚早期的造血過程，所以在本篇論文中利用TNAP MO顯微注射到斑馬魚的胚胎裡，使TNAP 剔除，TNAP基因KO之後斑馬魚的紅血球生成受到抑制，巨噬細胞的數量增加並且顆粒較大，嗜中性球的數量減少，所以我們懷疑這些巨噬細胞可能是分化不完全的狀態，因此我們想要了解斑馬魚早期巨噬細胞分化過程中相關基因的調控，所以利用巨噬細胞中早期的相關特異性基因進行PCR實驗，另外為了瞭解在TNAP缺乏的狀態下斑馬魚對LPS的耐受性，我們在施打MO後48小時給予不同濃度的LPS來觀察斑馬魚胚胎的存活率，如我們所預期的，TNAP缺乏的斑馬魚對LPS的抵禦能力明顯的下降許多，PCR的結果發現斑馬魚早期巨噬細胞相關的基因在TNAP缺乏的狀況下表現量與WT相比都有顯著的上升，證明了巨噬細胞在分化的過程中仍停留在早期未成熟的巨噬細胞階段，因此TNAP的缺乏在斑馬魚早期巨噬細胞分化過程中扮演著重要的角色。

關鍵字

巨噬細胞；鹼性磷酸酶早期造血； LPS

並列摘要

There are two types of alkaline phosphatase in zebrafish, intestinal alkaline phosphatase( IAP) and tissue nonspecific alkaline phosphatase( TNAP), in previous studies had been found that TNAP primary involved the early embryonic development of zebrafish it is known that TNAP was highly expressed on early development stage of zebrafish, especially at kidney, and kidney is also the major hematopoiesis organ in zebrafish. In previous studies, the lacked of TNAP would affect zebrafish early hematopoiesis process. In our study, we use TNAP MO to knockdown TNAP mRNA expression by microinjection to zebrafish embryo, and observed the number of macrophages is increased, the number of neutrophils is decreased, and erythropoiesis is suppressed. Therefore, we suspect that these macrophages may be stay in incomplete differentiation state. In the present study, we want to elucidate the role of TNAP on zebrafish early stage macrophages differentiation. We use macrophage specific makers to compare “TNAP deficient” with “wild type” by PCR analysis with different time course(12 hpf~72 hpf). To realize the LPS tolerance of macrophage, after injection TNAP MO, we treat LPS at 48 hpf with different consentration to observed zebrafish survival rate. As our expect, TNAP knockdown significantly reduced the resilience of macrophages. According to PCR result, the expression of macrophages associated gene were increased(TNAP MO), but the differentiation of macrophage was not completely. So, TNAP truly affect the differentiation of macrophages at early developmental stage of zebrafish.

並列關鍵字

marcrophages ； LPS ； early stage hematopoiesis ； TNAP

參考文獻

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鹼性磷酸酶在斑馬魚的巨噬細胞分化過程中扮演的角色

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