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研究生(外文):Chih Ming
論文名稱:在酵母菌內建立老鼠抑制食慾激素pancreatic polypeptide的表現系統
論文名稱(外文):The establishment of Saccharomyces cerevisiae expressive system of the rat hormone pancreatic polypeptide (PP) able to inhibited appetite
指導教授(外文):Rong-Tzong Tsai
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胰多肽(pancreatic polypeptide; PP)在攝取食物之後從胰臟小島(pancreatic islets)分泌及釋放進入循環系統。PP可能與體內能量平衡的調節及肥胖病源學方面有關,在臨床上已證實改變PP的分泌作用會影響進食行為,如肥胖疾病的個體,可以觀察到PP餐後的分泌較少。短暫的厭食效應(anorexigenic effects)是由於進食後大量PP釋放所造成。PP會導致負面的能量平衡,經由抑制食物的攝取及胃的排空,這個PP的作用機制包含調控腦下視丘中調節飲食胜肽的改變及迷走神經和迷走-交感神經反射弧活動的改變。
以前的文獻使用pancreatic polypeptide(PP)激素抑制食慾都是利用中樞或是周邊注射給予,而本次實驗是利用酵母菌內建立胰多肽的表現系統,我們希望將此由體內自然形成、完全無副作用的荷爾蒙,開發成口服的使用方式,尋求得到相同抑制食慾的效果。首先我們使用PCR的技術將胰多肽重組基因製造出來並轉殖入pRS406ΔA的質體中。接著,以限制酶將含有重組胜肽的PP切下轉殖到pET28載體中,獲得pET28-PP,接著在大腸桿菌中大量表達及純化PP重組胜肽。然後,再將包含完整PP重組基因以PCR方式從pET28-PP上面將其轉殖到酵母菌(Saccharomyces cerevisiae)表現載體pRS424-Gal中,接著,在酵母菌內建立老鼠抑制食慾激素pancreatic polypeptide的表現系統。

Pancreatic polypeptide (PP) is one such molecule, secreted from pancreatic islets and released into the circulation after ingestion of food. Altered PP secretion has been reported in clinical syndromes associated with abnormal eating behavior in humans. Blunted postprandial responses of PP have also been observed in individuals with morbid obesity, whereas subjects with anorexia nervosa are characterized by an exaggerated postprandial release of PP. These observations indicate that PP could be involved in the regulation of energy balance and the etiology of obesity. PP administration induces negative energy balance by suppressing food intake and gastric emptying. The mechanism of PP actions involves the changes in the expression of hypothalamic feeding-regulatory peptides and the activity of the vago-vagal and vago-sympathetic reflex arc.
According to previous documents, the main ways of using PP hormone to inhibit appetite are giving injection into central or peripheral administration. Whereas we want to construct a yeast strain which can express the PP in the cell, and the consumers can directly eat such kind of yeast to aquire the PP hormone. We hope to develop the method by eating the hormone naturally existed in human body and expect it to be without side effect. Firstly, we used the PCR technique to prepare a PP recombinant gene (about 135bps) in pRS406ΔA vector of Escherichia coli (the resulting plasmid was called pRS406ΔA-PP). Then, we cut the PP recombinant peptide gene by restriction enzyme and transit it to pET28 vector to create the plasmid pET28-PP. Subsequently, we produce and purify PP recombination peptide from E. coli. Afterward, we use PCR method to transit the recombinant genes that contain complete PP from the upper pET28-PP to Saccharomyces cerevisiae expressing vector pRS424-Gal to create the plasmid pRS424-Gal-PP.
In future, the ability of purified recombination PP and recombination PP 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 PP- containing saccaromycete into biological capsule.

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