葉酸是水溶性的維他命之一。在人體內中各種生理功能像是核甘酸、胺基酸以及 S-腺苷甲硫氨酸的合成都需要葉酸的存在。而且對於體內去氧核糖核酸、核糖核酸的甲基化扮演著很重要的角色。而葉酸缺乏則是會導致像是神經管缺陷等新生兒先天性的疾病，甚至在成人中，葉酸缺乏也會使得得到心血管疾病以及癌症的風險上升。在之前的研究報導指出在飲食中所含的葉酸會幫助預防肺的癌病變的產生。但是對於葉酸在肺的生理功能以及病理學上的角色則是尚待釐清。在我們的研究中，我們在先前實驗室所建利的斑馬魚葉酸缺乏的動物模式裡，我們發現葉酸缺乏會導致魚鰾的發育有所受損，而魚鰾更是在之前被報導在演化上哺乳類動物的肺的同源器官，我們發現有直到受精後第五天有將近九成的魚鰾沒有辦法正常發育，而這樣的現象是可以利用補充葉酸來回復的。而核糖核酸全胚胎原位雜交的結果中我們發現魚鰾中在受精後第三天時各個組織層的早期發育是沒有受到影響的，而這樣的結果也指出葉酸缺乏是影響了在之後的發育階段。此外我們發現了葉酸缺乏會導致胚胎半胱胺酸蛋白脢L的上升以及胱蛋白B 的下降，所以我們懷疑是不是因為如此而造成魚鰾的细胞外基质的受損而造成魚鰾無法正常的充氣膨脹。另外我們也對這兩種蛋白質的特性作探討分析，斑馬魚胱蛋白B 以及半胱胺酸蛋白脢L 都表現出與哺乳類相像的特性。而我們也純化出胱蛋白B，並且可以成功抑制木瓜蛋白酶的活性，而半胱胺酸蛋白脢L 的純化也正在進行當中。此外我們也看到半胱胺酸蛋白脢L 在胚胎早期會表現在斑馬魚的孵化線以及魚鰾上而胱蛋白B 則是在早期會均勻的表現在細胞中，而晚期則是會表現在斑馬魚心臟、血管以及魚鰾。在最後我們嘗試利用微注射的技術將純化好的胱蛋白B 打進三天大小魚中的魚鰾來進行治療，而結果顯示這樣子是可以成功的降低魚鰾受損的情形，也同時證明了在葉酸缺乏的斑馬魚中所看到的魚鰾受損是由於半胱胺酸蛋白脢L的上升以及胱蛋白B 的下降所導致。

Folate, a water-soluble vitamin, is essential for the biosynthesis of nucleotides, amino acid and S-adenosylmethionine, the major methyl-donor for most methylation reactions in cells. Folate deficiency (FD) causes embryonic defects and diseases including neural tube defect, megaloblastic anemia, cardiovascular disorders and cancers. Dietary folate was reported to protect individuals
against lung carcinogenesis. However, study on both physiological and pathological effects of folate
in lung is still limited. In the current study, we investigate the effects of folate deficiency on the
development of swimbladder, the evolutionary homolog of mammalian lung in zebrafish, using a transgenic line displaying inducible folate deficiency. We found that the swimbladder was absent in approximately 50% of FD embryos at 5 day-post-fertilization (dpf), which was partially reversed by folate supplementation. The results of Whole-mount in situ hybridization (WISH) with probes specific to developing swimbladder indicated a successful formation of swimbladder in FD embryos at early stages, suggesting a post-formation defect. The microarray data obtained from 5-dpf FD
embryos revealed an increased expression of cathepsin L, a lysosomal enzyme with strong elastinolytic activity, and a down-regulation of cystatin b, the inhibitor of cathepsin L in vivo. The up-regulation of cathepsin L was also reversed by folate supplement, supporting the causal link among folate deficiency, up-regulated cathepsin L and disappeared swimbladder. The distribution of cathepsin L mRNA examined by WISH was focused in embryonic hatch gland and swimbladder; whereas the cystatin b transcript was evenly distributed in zebrafish embryos before 24 hour-postfertilization and became focused in the heart and swimbladder of embryos after 1 dpf. The recombinant cystatin b was purified and shown to be structurally and functionally similar to its mammalian orthologs. The recombinant cathepsin L was successfully induced and undergoing
purification. Finally, we used microinject technique to send the recombinant CytB into the 3 dpf swimbladder chamber. And successfully partially rescue the swimbladder defect. In conclusion, our results suggested that the up-regulated cathepsin L and down-regulated cystatin b contributed to the
post-formation defects of zebrafish swimbladder observed in the FD embryos.

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