( 您好!臺灣時間:2024/06/20 23:37
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::


論文名稱:在酵母菌內建立老鼠抑制食慾激素Peptide YY (PYY)的表現系統
論文名稱(外文):The establishment of Saccharomyces cerevisiae expressive system of the rat hormone peptide YY (PYY) able to inhibit appetite
指導教授(外文):Rong-Tzong Tsai
  • 被引用被引用:0
  • 點閱點閱:3671
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
Peptide tyrosine-tyrosine(PYY)為胰多肽蛋白家族的成員之一,分佈起源於迴腸末端、結腸和直腸,是由36個胺基酸所組成的短鏈多肽物質,由文獻中已經證實,中樞或周邊系統輸注腸道荷爾蒙酪酪肽(PYY)可以抑制食物的攝取,減輕體重。當食縻進入腸道末端時,L細胞受到營養物的刺激,先將此訊號透過迷走神經傳入到達食慾的調節中樞,PYY荷爾蒙也會經由血液運送穿過血腦障壁至下視丘弓狀核,與Neuropeptide Y/Agouti-related peptide (NPY/AgRP)神經元的Y2 receptor結合,抑制NPY神經肽的釋放,相對的促進Pro-opiomelanocortin(POMC)神經元釋放出α-MSH,α-MSH隨即作用於M3/M4接受器(melanocortin receptor)達到抑制食慾的效果。
根據文獻已知PYY對於食慾抑制的臨床測試,在囓齒動物和人類中大都是使用注射的方式,而我們希望將此由體內自然形成、幾無副作用的荷爾蒙開發成口服的使用方式。首先我們使用PCR的技術將含有限制酶切位的PYY重組基因製造出來並轉殖入pRS406ΔA的質體中,利用colony PCR挑選含有pRS406ΔA-PYY的可能菌株,並且進行DNA定序,確認其序列的正確性。接著,以限制酶將含有重組基因的PYY切下轉殖到pET28載體中,獲得pET28-PYY,接著在大腸桿菌中大量表達及純化PYY重組胜肽。然後,再將N端含有His蛋白標籤的完整PYY重組基因以PCR方式從pET28-PYY上面將其轉殖到酵母菌(Saccharomyces cerevisiae)表現載體pRS424-Gal中,成功建構出pRS424-Gal-PYY表現質體。

The gut hormone Peptide YY (PYY) is a member of PP-fold peptide family. PYY is secreted from entero-endocrine L-cells and so named due to the presence of a tyrosine residue at each terminus of the polypeptide(36 amino acid). When dissolved food enter the end of the intestine tract, L-cell, subjecting to the stimulation of nutrients, would transmit the signal through vagal afferent to the appetite regulating center. Also, L-cell can produce PYY hormone. This hormone will flow with the blood, pass through blood-brain barrier (BBB), conduct to hypothalamic arcuate nucleus, bind to Y2 receptor, and inhibit the secretion of NPY/AgRP neuropeptide. Then, Pro-opiomelanocortins(POMC) neurons would release α-MSH that bind to M3/M4 receptor (melanocortin-3 receptor) and result to appetite inhibition.
According to the documents about PYY focusing on the clinical trials to inhibit appetite, and we found that injection is generally used as the method of clinical trials on rodents and human. However, we hope to develop the method by eating the hormone naturally formed from body and expect it to be little side-effect. We used the PCR technique to prepare a PYY recombinant gene (about 135bps). Next, the PYY recombinant gene was cloned into pRS406ΔA vector to construct the pRS406ΔA-PYY plasmid. After sequencing, We confirm PYY gene. Rstriction enzymes cut pRS406ΔA-PYY plasmid at very specific locations and we prepare the insert. Cloning PYY recombinant gene into pET28 vector of Escherichia coli (the resulting plasmid was called pET28-PYY). The recombinant pET28-PYY was transformed into E. coli (BL21 cell). Then, we perform Ni-column purification of recombinant PYY. The His-PYY were confirmed using Western bolt analysis with anti-His antibodies. Moreover, we would use PCR technique to transfer the recombinant gene, containing complete PYY, from pET28-PYY to yeast expressing vector pRS424-Gal.We have constructed the yeast expressive plasmid , pRS424-Gal-PYY.
In future, the ability of purified recombinant PYY and recombinant PYY expressing yeasts to inhibit rat appetite will be assessed by injection or eating respectively. These results will form the basis for the assessment of developing saccaromycete into biological capsule.


Asakawa, A., Inui, A., Kaga, T., Yuzuriha, H., Nagata, T., Ueno, N., Makino, S., Fujimiya, M., Niijima, A., Fujino, M.A., and Kasuga, M. (2001). Ghrelin is an appetite-stimulatory signal from stomach with structural resemblance to motilin. Gastroenterology. 120, 337-45.
Anubhuti, A.S. (2006). Role of neuropeptides in appetite regulation and obesity. Neuropeptides. 40, 375-401.
Astrup, A. (2005). Effect of rimonabant on weight reduction and cardiovascular risk. Lancet. 366, 368.
Batterham, R.L., Cowley, M.A., Small, C.J., Herzog, H., Cohen, M.A., Dakin, C,L., Wren, A.M., Brynes, A.E., Low, M.J., Ghatei, M.A., Cone, R.D., and Bloom, S.R. (2002). Gut hormone PYY(3-36) physiologically inhibits food intake. Nature. 418, 650-654.
Batterham, R.L., Heffron, H., Kapoor, S., Chivers, J.E, Chandarana, K., Herzog, H., Le Roux, C.W., Thomas, E.L., Bell, J.D., and Withers, D.J. (2006). Critical role for peptide YY in protein-mediated satiation and body-weight regulation. Cell Metab. 4, 223-33.
Batterham, R.L., Cohen, M.A., Ellis, S.M., Le Roux, C.W., Withers, D.J., Frost, G.S., Ghatei, M.A., and Bloom, S.R. (2003). Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med. 349, 941-8.
Batterham, R.L., Le Roux, C.W., Cohen, M.A., Park, A.J., Ellis, S.M., Patterson, M., Frost, G.S., Ghatei, M.A., and Bloom, S.R. (2003). Pancreatic polypeptide reduces appetite and food intake in humans. J. Clin. Endocrinol. Metab. 88, 3989–3992.
Bowne, W.B., Julliard, K., Castro, A.E., Shah, P., Morgenthal, C.B., and Ferzli, G.S. (2006). Laparoscopic gastric bypass is superior to adjustable gastric band in super morbidly obese patients: a prospective, comparative analysis. Arch Surg.141, 683-9.
Bewick, G.A., Gardiner, J.V., Dhillo, W.S., Kent, A.S., White, N.E., Webster, Z., Ghatei, M.A., and Bloom, S.R. (2005). Post-embryonic ablation of AgRP neurons in mice leads to a lean, hypophagic phenotype. FASEB J. 19, 1680-1682.
Barsh, G.S., Farooqi, I.S., and O''Rahilly, S. (2000). Genetics of body-weight regulation. Nature. 404, 644-51.
Baggio, L.L., Huang, Q., Brown, T.J., and Drucker, D.J. (2004). Oxyntomodulin and glucagon-like peptide-1 differentially regulate murine food intake and energy expenditure. Gastroenterology. 127, 546–558.
Cowley, M.A., Cone, R.D., Enriori, P., Louiselle, I., Williams, S.M., and Evans, A.E. (2003). Electrophysiological actions of peripheral hormones on melanocortin neurons. Ann. N. Y. Acad. Sci. 994,175–186.
Challis, B.G., Coll, A.P., Yeo, G.S., Pinnock, S.B., Dickson, S.L., Thresher, R.R., Dixon, J., Zahn, D., Rochford, J.J., White, A., Oliver, R.L., Millington, G., Aparicio, S.A., Colledge, W.H., Russ, A.P., Carlton, M.B., and O''Rahilly, S. (2004). Mice lacking pro-opiomelanocortin are sensitive to high-fat feeding but respond normally to the acute anorectic effects of peptide-YY(3-36). Proc Natl Acad Sci USA. 101, 4695-4700.
Cohen, M.A., Ellis, S.M., Le Roux, C.W., Batterham, R.L., Park, A., Patterson, M., Frost, G.S., Ghatei, M.A., and Bloom, S.R. (2003). Oxyntomodulin suppresses appetite and reduces food intake in humans. J Clin Endocrinol Metab. 88, 4696–4701.
Drueker, D.J. (2006). The biology of ineretin hormones. Cell Metab. 3, 153–165.
Dakin, C.L., Gunn, I., Small, C.J., Edwards, C.M., Hay, D.L., Smith, D.M.,Ghatei, M.A., and Bloom, S.R. (2001). Oxyntomodulin inhibits food intake in the rat. Endocrinology. 142, 4244–4250.
Dockray, G.J. (2009). Cholecystokinin and gut-brain signalling. Regul Pept. 155, 6-10.
Eberlein, G.A., Eysselein, V.E., Schaeffer, M., Layer, P., Grandt, D., Goebell, H., Niebel, W., Davis, M., Lee, T.D., and Shively, J.E. (1989). A new molecular form of PYY: structural characterization of human PYY(3- 36) and PYY(1-36). Peptides. 10, 797-803.
Esposito, K., and Giugliano, D. (2005). Effect of rimonabant on weight reduction and cardiovascular risk. Lancet. 366, 367-368.
Foxcroft, D.R., and Milne, R. (2000). Orlistat for the treatment of obesity: rapid review and cost effectiveness model. Obes. 1, 121–126.
Field, B.C., Wren, A.M., Cooke, D., and Bloom, S.R. (2008). Gut hormones as potential new targets for appetite regulation and the treatment of obesity. Drugs. 68, 147-163.
Finer, N. (2002). Sibutramine: its mode of action and efficacy. Int J Obes Relat Metab Disord. 26, S29–S33.
Fujimiya, M., and Inuib, A. (2000). Peptidergic regulation of gastrointestinal motility in rodents. Peptides. 21, 1565-1582.
Greeley, G.H. Jr., Jeng, Y.J., Gomez, G., Hashimoto, T., Hill, F.L., Kern, K., Kurosky, T., Chuo, H.F., and Thompson, J.C. (1989). Evidence for regulation of peptide-YY release by the proximal gut. Endocrinology. 124, 1438-43.
Hirschel B. (2005). Effect of rimonabant on weight reduction and cardiovascular risk. Lancet. 366, 369-370.
Holst, J.J. (2004). On the physiology of GIP and GLP-1. Horm Metab Res. 36, 747–754.
Hameed, S., Dhillo, W.S., and Bloom, S.R. (2009). Gut hormones and appetite control. Oral Diseases. 15, 18–26.
Ioannides-Demos, L.L., Proietto, J., and McNeil, J.J. (2005). Pharmacotherapy for obesity. Drugs. 65, 1391-418.
Kojima, M., Hosoda, H., Date, Y., Nakazato, M., Matsuo, H., and Kangawa, K. (1999). Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature. 402, 656-660.
Kuo, L.E., Kitlinska, J.B., Tilan, J.U., Li, L., Baker, S.B., Johnson, M.D., Lee, E.W., Burnett, M.S., Fricke, S.T., Kvetnansky, R., Herzog, H., and Zukowska, Z. (2007). Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome. Nat Med. 13, 803-811.
Kopelman, P.G. (2000). Obesity as a medical problem. Nature. 404, 635-643.
Keire, D.A., Mannon, P., Kobayashi, M., Walsh, J.H., Solomon ,T.E., and Reeve, J.R. Jr. (2000). Primary structures of PYY, [Pro(34)]PYY, and PYY-(3-36) confer different conformations and receptor selectivity. Am J Physiol Gastrointest Liver Physiol. 279, 126-131.
Larhammar, D. (1996). Structural diversity of receptors for neuropeptideY, peptide YY and pancreatic polypeptide. Regul Pept. 65,165–174.
Le Roux, C.W., Aylwin, S.J, Batterham, R.L., Borg, C.M., Coyle, F., Prasad, V., Shurey, S., Ghatei, M.A., Patel, A.G., and Bloom, S.R.(2006). Gut hormone profiles following bariatric surgery favor an anorectic state, facilitate weight loss, and improve metabolic parameters. Ann Surg. 243,108-114.
Le Roux, C.W., and Bloom, S.R. (2005). Peptide YY, appetite and food intake. Proc Nutr Soc. 64, 213-6.
Murray, C.D., Le Roux, C.W., Gouveia, C., Bassett, P., Ghatei, M.A., Bloom, S.R., Emmanuel, A.V., and Gabe, S.M. (2006). The effect of different macronutrient infusions on appetite, ghrelin and peptide YY in parenterally fed patients. Clin Nutr. 25, 626-633.
Martin, N.M., Small, C.J., Sajedi, A., Patterson, M., Ghatei, M.A., and Bloom, S.R. (2004). Pre-obese and obese agouti mice are sensitive to the anorectic effects of peptide YY3-36 but resistant to ghrelin. Intern J Obes. 28, 886-893.
Meeran, K., O’Shea, D., Edwards, C.M., Turton, M.D., Heath, M.M., Gunn, I., Abusnana, S., Rossi, M., Small, C.J., Goldstone, A.P., Taylor, G.M., Sunter, D., Steere, J., Choi, S.J., Ghatei, M.A., and Bloom, S.R. (1999). Repeated intracerebroventricular administration of glucagon-like peptide-1-(7–36) amide or exendin-(9–39) alters body weight in the rat. Endocrinology. 140, 244–250.
Moran, T.H., and Schwartz, G.J. (1994). Neurobiology of cholecystokinin.Crit. Rev. Neurobiol. 9, 1–28.
Morton, G.J., Cummings, D.E., Baskin, D.G., Barsh, G.S., and Schwartz, M.W. (2006). Central nervous system control of food intake and body weight. Nature. 443, 289-295.
Oesch, S., Rjiuegg, C., Fischer, B., Degen, L., and Beglinger, C. (2006). Effect of gastric distension prior to eating on food intake and feelings of satiety in humans. Physiol Behav. 87, 903-10.
Pittner, R.A., Moore, C.X., Bhavsar, S.P., Gedulin, B.R., Smith, P.A., Jodka, C.M., Parkes, D.G., Paterniti, J.R., Srivastava, V.P., and Young, A.A. (2004). Effects of PYY[3–36] in rodent models of diabetes and obesity. International Journal of Obesity. 28, 963–971.
Poindexter, G.S., Bruce, M.A., LeBoulluec, K.L., Monkovic, I., Martin, S.W., Parker, E.M., Iben, L.G., McGovern, R.T., Ortiz, A.A., Stanley, J.A., Mattson, G.K., Kozlowski, M., Arcuri, M., and Antal-Zimanyi, I. (2002). Dihydropyridine neuropeptide Y Y(1) receptor antagonists. Bioorg Med Chem Lett. 12, 379-82.
Raposinho, P.D., Pierroz, D.D., Broqua, P., Whitea, R.B., Pedrazzini, T., and Aubert, M.L. (2001). Chronic administration of neuropeptide Y into the lateral ventricle of C57BL/6J male mice produces an obesity syndrome including hyperphagia, hyperleptinemia, insulin resistance, and hypogonadism. Mol Cell Endocrinol. 185, 195-204.
Stock, M.J. (1997). Sibutramine: a review of the pharmacology of a novel anti-obesity agent. Int J Obes Relat Metab Disord. 21, S25–S29.
Schwartz, M.W., Woods, S.C., Porte, D. Jr., Seeley, R.J., and Baskin, D.G. (2000). Central nervous system control of food intake. Nature. 404, 661–671.
Sainsbury, A., Schwarzer, C., Couzens, M., Fetissov, S., Furtinger, S., Jenkins, A., Cox, H.M., Sperk, G., Hökfelt, T., and Herzog, H. (2002). Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice. Proe Natl Acad Sci USA. 99, 8938-43.
Schmidt, P.T., Näslund, E., Grybäck, P., Jacobsson, H., Holst, J.J., Hilsted, L., and Hellström, P.M. (2005). A role for pancreatic polypeptide in the regulation of gastric emptying and short-term metabolic control. Clinical Endocrinology and Metabolism 90, 5241–5246.
Small, C.J., and. Bloom, S.R. (2004). Gut hormones and the control of Appetite. Trends Endocrinol Metab.15, 259-63.
Tatemoto, K., and Mutt, V. (1980). Isolation of two novel candidate hormones using a chemical method for finding naturally occurring polypeptides. Nature. 285, 417-418.
Ueno, N., Inui, A., Iwamoto, M., Kaga, T., Asakawa, A., Okita, M., Fujimiya, M., Nakajima, Y., Ohmoto, Y., Ohnaka, M., Nakaya, Y., Miyazaki, J.I., and Kasuga, M. (1999). Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice. Gastroenterology 117,1427–1432.
Van, O.B., Killestein, J., and Polman, C.h. (2005). Effect of rimonabant on weight
reduction and cardiovascular risk. Lancet. 366, 368–369.
Vergoni, A.V., and Bertolini, A. (2000). Role of melanocortins in the central control of feeding. Eur J Pharmacol. 405, 25-32.
Wren, A.M., and Bloom, S.R. (2007). Gut hormones and appetite control.Gastroenterology. 132, 2116–2130.
Wynne, K., Park, A.J., Small, C.J., Meeran, K ., Ghatei, M.A., Frost, G.S., and Bloom, S.R. (2006). Oxyntomodulin increases energy expenditure in addition to decreasing energy intake in overweight and obese humans: a randomised controlled trial. International Journal of Obesity. 30, 1729–1736.

第一頁 上一頁 下一頁 最後一頁 top