臺灣博碩士論文加值系統

鋅為生物體必需微量營養素，是構成轉錄因子之必要組成而調節基因表現。鋅缺乏時以食慾不振為最早出現的症狀，而已知腸道也會參與食慾的調控。因此本實驗針對缺鋅大鼠，利用高通量（high-throughput）DNA微陣列分析技術，以全面性分析缺鋅對小腸基因表現的影響，並嘗試發現缺鋅影響食慾的腸道因子。實驗動物為Wistar品系公鼠，分控制組、缺鋅組、中度缺鋅組及缺鋅對飼育組；中度缺鋅組於第19天改餵食缺鋅飼料（MD）及正常飼料（MPF）5天。各組於餵養24天後犧牲，然後收取小腸組織，萃取小腸黏膜total RNA，首先以pooled樣品進行DNA微陣列實驗分析，並以Q-PCR驗證有差異之特定基因。缺鋅組之體重、攝食量、飼料利用率、肝臟及血漿鋅含量都顯著低於控制組；對飼育組之飼料利用率、肝臟及血漿鋅濃度則高於缺鋅組。由微陣列分析結果顯示，與控制組比較，缺鋅組之基因表現升為2倍以上的基因有90個，表現降為50％以下之基因有84個，總有174個表現差異基因；對飼育組之表現升為2倍以上的基因有47個，表現降為50％以下之基因有29個，共76個表現差異基因。比較缺鋅與對飼育組可見，缺鋅改變之基因數較限食多，兩組只有20個基因表現是變化相同，其餘皆不同，表示缺鋅對基因的影響具有獨特性，並非透過限食作用。缺鋅組之metallothionein與ZnT-2基因表現減少，只有控制組的0.28倍與0.41倍，而neuromedin U則增加，為控制組的2.76倍；對飼育組則沒有這些變化。以Q-PCR進行大鼠個別驗證可見，缺鋅鼠之neuromedin U基因表現與缺鋅時攝食波動趨勢相關，已知neuromedin U在腦部有抑制食慾的作用，故推測neuromedin U與缺鋅對於食慾的影響有關。本研究首度發現鋅營養狀態可改變neuromedin U之基因表現，有助於探討缺鋅對食慾的影響。

Zinc is an indispensable metal nutrient and involved in regulation of gene expression since it is an essential component of the zinc finger structure of many transcription factors. One of the early zinc deficiency symptoms is depressed food intake, which can be regulated by both central nervous and peripheral neuroendocrine systems including the gastrointestinal tract. Therefore, we used a high-throughput, whole genome rat DNA microarray assay to analyze the gene expression profile of the small intestinal in zinc deficient rat, and tried to identify novel intestinal factor for appetite control by zinc. Twenty Wistar male rats were divided into four groups: a zinc-deficient group (ZnD, zinc < 2 ppm), a pair-fed group (PZnD, zinc = 50 ppm), a marginal zinc-deficient group (MZnD, zinc = 5 ppm), and a zinc-adequate group (ZnA, zinc = 50 ppm). All groups were fed with corresponding diet for 24 days except the MZnD, in which 3 rats were changed to the zinc deficient diet and two to the pair-fed diet for the last 5 five days. Total RNA of small intestinal mucosa was extracted from each rat, pooled RNA samples corresponding to ZnD, PZnD and ZnA were subjected to Agilent Rat V2 Oligo Microarray assay. Zn deficient rats had significantly lower body weight, food intake, feed efficiency and zinc content in liver and plasma. Between ZnD and ZnA groups, a total of 174 genes were differentially expressed, with 90 genes one-fold higher and 84 genes 50% lower in the ZnD group; whereas between PZnD and ZnA groups, a total of 76 genes were differentially expressed, with 47 genes one-fold higher and 29 genes 50% lower in the PZnD group. Between ZnD and PZnD groups, only 20 genes were similarly changed, indicating that zinc deficiency exerts a specific effect other than food restriction on intestinal gene expression. In addition to metallothionein and zinc transporter ZnT-2, neuromedin U expression (NmU) was specifically altered by zinc deficiency and increased to 2.76-fold. NmU expression of individual rat was measured with quantitative RT-PCR and showed an association with zinc status that NmU expression can be linked to the cyclical pattern of food intake in the ZnD group. In conclusion, we first report a novel relationship between zinc nutrition and intestinal neuromedin U expression, which may help to elucidate the appetite regulation modulated by zinc deficiency.

中文摘要1
英文摘要2
第一章 文獻回顧 9
第一節 鋅 9
一、鋅的生化功能 9
二、鋅缺乏的臨床症狀 10
三、鋅與食慾的關係 10
第二節 食慾的調節 12
一、中樞神經系統 12
二、周邊組織系統 12
三、Neuromedin U 14
第三節 微陣列分析技術 17
第四節 生物資訊分析軟體 17
第五節 研究動機與目的 18

第二章 材料與方法 19
第一節 動物實驗 19
一、實驗設計 19
二、動物飼養 19
三、飼料組成及製備 20
第二節 動物犧牲及樣品收集 21
第三節 小腸黏膜組織total RNA萃取 21
第四節 微陣列分析實驗（Microarray experiment）22
一、實驗設計 22
二、實驗流程 23
第五節 生物資訊分析 23
一、GeneSpring 23
二、ArrayXPath 24
三、Ingenuity pathway analysis 24
第六節 即時定量聚合酶連鎖反應（Real-Time Quantitative Polymerase Chain Reaction, QRT-PCR）24
第七節 定量飼料、血漿及肝臟鋅濃度 26
第八節 統計分析 26

第三章 結果 28
第一節 建立小腸黏膜total RNA萃取 28
第二節 缺鋅動物之生長狀況 29
一、體重與攝食量 29
二、組織重量與組織相對重量 29
三、肝臟與血漿之鋅含量 30
第三節 缺鋅與限食對小腸基因之影響 30
一、基因表現差異 30
二、生物錄進與功能 31
三、轉錄因子基因表現 32
四、NmU基因表現變化 32

第四章 討論與結論 69
第一節 實驗方法、工具檢討 69
一、小腸黏膜RNA萃取方法改良 69
二、生物資訊分析軟體 69
第二節 微陣列結果 70
第三節 NmU在鋅缺乏時可能扮演的角色 71

第五章 參考文獻 74

附錄 78
一、RNA品質確認-Bioanalyzer分析結果 78
二、RNA品質確認-Bioanalyzer分析圖譜 79

Auld DS (2001) Zinc coordination sphere in biochemical zinc sites. Biometals 14, 271-313.

Austin C, Oka M, Nandha KA, Legon S, Khandan-Nia N, Lo G & Bloom SR (1994) Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract. J Mol Endocrinol 12, 257-263.

Badman MK & Flier JS (2005) The gut and energy balance: visceral allies in the obesity wars. Science 307, 1909-1914.

Ballesta J, Carlei F, Bishop AE, Steel JH, Gibson SJ, Fahey M, Hennessey R, Domin J, Bloom SR & Polak JM (1988) Occurrence and developmental pattern of neuromedin U-immunoreactive nerves in the gastrointestinal tract and brain of the rat. Neuroscience 25, 797-816.

Beyersmann D & Haase H (2001) Functions of zinc in signaling, proliferation and differentiation of mammalian cells. Biometals 14, 331-341.

Blanchard RK & Cousins RJ (1996) Differential display of intestinal mRNAs regulated by dietary zinc. Proc Natl Acad Sci U S A 93, 6863-6868.

Blanchard RK, Moore JB, Green CL & Cousins RJ (2001) Modulation of intestinal gene expression by dietary zinc status: effectiveness of cDNA arrays for expression profiling of a single nutrient deficiency. Proc Natl Acad Sci U S A 98, 13507-13513.

Brighton PJ, Szekeres PG & Willars GB (2004) Neuromedin U and its receptors: structure, function, and physiological roles. Pharmacol Rev 56, 231-248.

Chesters JK & Quarterman J (1970) Effects of zinc deficiency on food intake and feeding patterns of rats. Br J Nutr 24, 1061-1069.

Chomczynski P & Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156-159.

Domin J, Ghatei MA, Chohan P & Bloom SR (1987) Neuromedin U--a study of its distribution in the rat. Peptides 8, 779-784.

Essatara MB, McClain CJ, Levine AS & Morley JE (1984) Zinc deficiency and anorexia in rats: the effect of central administration of norepinephrine, muscimol and bromerogocryptine. Physiol Behav 32, 479-482.
Flier JS (2004) Obesity wars: molecular progress confronts an expanding epidemic. Cell 116, 337-350.

Furness JB, Pompolo S, Murphy R & Giraud A (1989) Projections of neurons with neuromedin U-like immunoreactivity in the small intestine of the guinea-pig. Cell Tissue Res 257, 415-422.

Gaetke LM, Frederich RC, Oz HS & McClain CJ (2002) Decreased food intake rather than zinc deficiency is associated with changes in plasma leptin, metabolic rate, and activity levels in zinc deficient rats( small star, filled). J Nutr Biochem 13, 237-244.

Golub MS, Keen CL, Gershwin ME & Hendrickx AG (1995) Developmental zinc deficiency and behavior. J Nutr 125, 2263S-2271S.

Hanada R, Teranishi H, Pearson JT, Kurokawa M, Hosoda H, Fukushima N, Fukue Y, Serino R, Fujihara H, Ueta Y, Ikawa M, Okabe M, Murakami N, Shirai M, Yoshimatsu H, Kangawa K & Kojima M (2004) Neuromedin U has a novel anorexigenic effect independent of the leptin signaling pathway. Nat Med 10, 1067-1073.

Hanada T, Date Y, Shimbara T, Sakihara S, Murakami N, Hayashi Y, Kanai Y, Suda T, Kangawa K & Nakazato M (2003) Central actions of neuromedin U via corticotropin-releasing hormone. Biochem Biophys Res Commun 311, 954-958.

Hashimoto T, Masui H, Uchida Y, Sakura N & Okimura K (1991) Agonistic and antagonistic activities of neuromedin U-8 analogs substituted with glycine or D-amino acid on contractile activity of chicken crop smooth muscle preparations. Chem Pharm Bull (Tokyo) 39, 2319-2322.

Howard AD, Wang R, Pong SS, Mellin TN, Strack A, Guan XM, Zeng Z, Williams DL, Jr., Feighner SD, Nunes CN, Murphy B, Stair JN, Yu H, Jiang Q, Clements MK, Tan CP, McKee KK, Hreniuk DL, McDonald TP, Lynch KR, Evans JF, Austin CP, Caskey CT, Van der Ploeg LH & Liu Q (2000) Identification of receptors for neuromedin U and its role in feeding. Nature 406, 70-74.

Hughes TR, Mao M, Jones AR, Burchard J, Marton MJ, Shannon KW, Lefkowitz SM, Ziman M, Schelter JM, Meyer MR, Kobayashi S, Davis C, Dai H, He YD, Stephaniants SB, Cavet G, Walker WL, West A, Coffey E, Shoemaker DD, Stoughton R, Blanchard AP, Friend SH & Linsley PS (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat Biotechnol 19, 342-347.

Jethwa PH, Small CJ, Smith KL, Seth A, Darch SJ, Abbott CR, Murphy KG, Todd JF, Ghatei MA & Bloom SR (2005) Neuromedin U has a physiological role in the regulation of food intake and partially mediates the effects of leptin. Am J Physiol Endocrinol Metab 289, E301-305.

Kangawa K, Minamino N, Fukuda A & Matsuo H (1983) Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord. Biochem Biophys Res Commun 114, 533-540.

Kojima M, Haruno R, Nakazato M, Date Y, Murakami N, Hanada R, Matsuo H & Kangawa K (2000) Purification and identification of neuromedin U as an endogenous ligand for an orphan receptor GPR66 (FM3). Biochem Biophys Res Commun 276, 435-438.

Konturek SJ, Konturek JW, Pawlik T & Brzozowski T (2004) Brain-gut axis and its role in the control of food intake. J Physiol Pharmacol 55, 137-154.

Korner J & Leibel RL (2003) To eat or not to eat - how the gut talks to the brain. N Engl J Med 349, 926-928.

Krebs NE & Hambidge KM (2001) Zinc metabolism and homeostasis: the application of tracer techniques to human zinc physiology. Biometals 14, 397-412.

Lee RG, Rains TM, Tovar-Palacio C, Beverly JL & Shay NF (1998) Zinc deficiency increases hypothalamic neuropeptide Y and neuropeptide Y mRNA levels and does not block neuropeptide Y-induced feeding in rats. J Nutr 128, 1218-1223.

Lo G, Legon S, Austin C, Wallis S, Wang Z & Bloom SR (1992) Characterization of complementary DNA encoding the rat neuromedin U precursor. Mol Endocrinol 6, 1538-1544.

Mantzoros CS, Prasad AS, Beck FW, Grabowski S, Kaplan J, Adair C & Brewer GJ (1998) Zinc may regulate serum leptin concentrations in humans. J Am Coll Nutr 17, 270-275.

Minamino N, Kangawa K & Matsuo H (1983) Neuromedin B: a novel bombesin-like peptide identified in porcine spinal cord. Biochem Biophys Res Commun 114, 541-548.

Minamino N, Kangawa K & Matsuo H (1985) Neuromedin U-8 and U-25: novel uterus stimulating and hypertensive peptides identified in porcine spinal cord. Biochem Biophys Res Commun 130, 1078-1085.

Moynahan EJ (1974) Letter: Acrodermatitis enteropathica: a lethal inherited human zinc-deficiency disorder. Lancet 2, 399-400.

Prasad AS (1991) Discovery of human zinc deficiency and studies in an experimental human model. Am J Clin Nutr 53, 403-412.

Rains TM & Shay NF (1995) Zinc status specifically changes preferences for carbohydrate and protein in rats selecting from separate carbohydrate-, protein-, and fat-containing diets. J Nutr 125, 2874-2879.

Rink L & Gabriel P (2000) Zinc and the immune system. Proc Nutr Soc 59, 541-552.

Shay NF & Cousins RJ (1993) Cloning of rat intestinal mRNAs affected by zinc deficiency. J Nutr 123, 35-41.

Sparmann G, Jaschke A, Loehr M, Liebe S & Emmrich J (1997) Tissue homogenization as a key step in extracting RNA from human and rat pancreatic tissue. Biotechniques 22, 408-410, 412.

Strader AD & Woods SC (2005) Gastrointestinal hormones and food intake. Gastroenterology 128, 175-191.

Thiers RE & Vallee BL (1957) Distribution of metals in subcellular fractions of rat liver. J Biol Chem 226, 911-920.

Vallee BL & Falchuk KH (1993) The biochemical basis of zinc physiology. Physiol Rev 73, 79-118.

van Breda SG, van Agen E, van Sanden S, Burzykowski T, Kienhuis AS, Kleinjans JC & van Delft JH (2005) Vegetables affect the expression of genes involved in anticarcinogenic processes in the colonic mucosa of C57BL/6 female mice. J Nutr 135, 1879-1888.

Wallwork JC & Sandstead HH (1983) Effect of zinc deficiency on appetite and free amino acid concentrations in rat brain. J Nutr 113, 47-54.

Wang Y, Wimmer U, Lichtlen P, Inderbitzin D, Stieger B, Meier PJ, Hunziker L, Stallmach T, Forrer R, Rulicke T, Georgiev O & Schaffner W (2004) Metal-responsive transcription factor-1 (MTF-1) is essential for embryonic liver development and heavy metal detoxification in the adult liver. Faseb J 18, 1071-1079.

Woods SC (2004) Gastrointestinal satiety signals I. An overview of gastrointestinal signals that influence food intake. Am J Physiol Gastrointest Liver Physiol 286, G7-13.