抗酶抑制因子減低細胞傷害物質對於斑馬魚胚胎發育之干擾

研究目的：抗酶抑制因子(Antizyme inhibitor, AZI)是細胞內調控鳥胺酸去羧化酶（Ornithine decarboxylase; ODC, EC 4.1.1.17）活性與多胺（polyamines）運送有關的物質，執行各種細胞內分子功能的各種角色，包括發育的調控、細胞週期、細胞生長與死亡、細胞分化與腫瘤增生。AZI與ODC蛋白序列以及分子量具有高度的相似性，抗酶抑制因子與抗酶(Antizyme, AZ)的結合能力比鳥胺酸脫羧酶還強，能夠回復鳥胺酸脫羧酶受抗酶抑制的情形。斑馬魚在胚胎發育的機制和哺乳類非常類似，並且具有體型小、飼養容易、世代週期短以及魚卵大而透明易於胚胎時期器官的觀察。然而對於AZI調控斑馬魚生理之研究尚未得到驗證。因此本實驗在細胞損傷之情形欲藉由調控AZI來探討AZI對於斑馬魚胚胎發育所扮演之角色。研究方法：藉由UVB以及H2O2、CQ、TG、VP16等化學的方式給予，使得斑馬魚細胞造成損傷。利用顯微注射AZI DNA或蛋白質來提高AZI之表現。藉由照相得知型態變化和TUNEL assay來確認細胞凋亡造成的DNA損傷程度和位置與RT-PCR檢視mRNA表現的差異。研究結果：我們成功建構斑馬魚的實驗模式。由實驗結果得知UVB、H2O2、CQ 會造成斑馬魚胚胎發育受到影響:畸胎(心臟發育不全、卵黃囊腫大、軀幹及尾部扭曲)、致死率提高、孵化率降低，細胞凋亡的情形也比較嚴重，並且使得AZI表現降低。在功能性分析中不論是給予AZI DNA或者是直接給予AZI蛋白質來提高AZI的表現都可以回復上述刺激所造成的傷害，此外施打AZI shRNA 也會提高胚胎致死率。結論：根據實驗結果，我們認為AZI對於外界刺激造成斑馬魚胚胎發育損傷具有修復性的功能。

BACKGROUD: Antizyme inhibitor (AZI) delicately regulates ornithine decarboxylase (ODC) enzyme activity and polyamine transport. ODC plays a numerous roles in molecular and cellular functions, including developmental regulation, cell cycle, proliferation, cell death, differentiation and tumorigenesis. AZI has similar sequence and molecular weight with ODC. AZI, which has higher binding affinity to antizyme (AZ) more than ODC, can rescue ODC from AZ inhibition. The developmental mechanisms of zebrafish are similar with mammalian, highly. They are tiny size, easy feeding, short generation period, big limpid eggs, and developmental organs observation easily. However, how does AZI participate in the zebrafish is still unidentified. Therefore, we evaluate the effect of AZI on cellular insults during zebrafish embryonic development.METHODS: Cellular insults such as UVB, H2O2 and CQ were treated in zebrafish embryonic development. AZI expression was enhanced by microinjection of AZI DNA or protein. The phenotypes were observed and photographed with a microscope equipped with a digital camera. TUNEL assay was applied to identify apoptosis. The expressions of AZI, ODC and AZ mRNA were analyzed by using RT-PCR.RESULTS: We constructed the experiment model, zebrafish. We shown that UVB, H2O2 and CQ induced zebrafish embryonic development abnormal: malformation (similar defects of brain heart, development and heart function was abnormal, yolk sac enlargement and contortion of trunk and tail), increased death rate, decreased hatch rate and apoptotic level raise. Furthermore, AZI expression was reduced in cellular insults. The overexpression of AZI could recover the injuries as mention above. Beside, AZI shRNA leads malformation and increase death rate in zebrafish.CONCLUSIONS: According to these data, we suggest that AZI could resist stress and recover injury in zebrafish embryonic development.