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National Chung Hsing University Institutional Repository - NCHUIR > 獸醫學院 > 獸醫學系所 > 依資料類型分類 > 碩博士論文 >  轉殖胰島素基因及幹細胞降低糖尿病大白鼠血糖之研究

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/155292

標題: 轉殖胰島素基因及幹細胞降低糖尿病大白鼠血糖之研究
Transfection of rat insulin gene with retrovirus vector and stem cell in diabetic rat
作者: 賴敏銓
Lai, Min-Chuan
Contributors: 楊繼
Chi Yang
獸醫學系暨研究所
關鍵字: 胰島素基因;幹細胞;糖尿病大白鼠
insulin gene;stem cell;diabetic rat
日期: 2013
Issue Date: 2013-11-21 11:49:51 (UTC+8)
Publisher: 獸醫學系暨研究所
摘要: 人類和動物的糖尿病患通常透過飲食控制及口服藥物(如:sulfonylurea)或注射胰島素予以治療,但是注射給藥有所不便,注射過量易造成血糖過低甚而致死,口服雖較方便安全,但將近有25-30 %患者無法達到滿意之控制,因此有必要發展其他可以改善人類及動物第1型糖尿病的方法。近年來,利用基因治療的方式,將胰島素基因轉殖入非胰島細胞,以合成胰島素成為治療糖尿病的方法之一,目前已知所使用之病毒載體有反轉錄病毒(Retrovirus)、Lentivirus、腺病毒(Adenovirus)、Adeno-associated virus及疱疹病毒(Herpes simplex virus)等成功轉殖入細胞或體內合成胰島素,但是使用上各有其優缺點。本研究之過程以選殖大白鼠胰島素Ⅰ基因全長,及其起動子(promoter)與促進子(enchancer),分別嵌入已構築好之反轉錄病毒載體後,轉殖入老鼠纖維母細胞中形成完整的病毒顆粒後,合成與表現大白鼠之胰島素,3天後以競爭性放射免疫法(radioimmunoassay)測定胰島素之含量。結果顯示反轉錄病毒表現載體確實能表現胰島素基因,其表現量最高可達到4806.35 ± 53.72 (pg/ml);實驗顯示含有起動子與促進子的胰島素基因,其胰島素的表現量比未包含起動子與促進子的胰島素基因高出61 %,可見胰島素起動子與促進子基因在表現系統中有其調控的重要性;同時將已完成轉殖胰島素基因之老鼠纖維母細胞注射入糖尿病大白鼠胰臟中,10天內糖尿病大白鼠血糖有明顯下降之趨勢;顯見利用反轉錄病毒載體轉殖胰島素基因可做為大白鼠體內或體外之治療模式。除此之外,研究顯示取代或更新糖尿病患者受損胰臟β細胞的療法,在未來是很有潛力的治療方式,目前已知的β細胞包括成熟的幹細胞與胚胎來源的幹細胞,成熟的幹細胞主要位於胰小管的上皮細胞及胰島細胞,胚胎來源幹細胞則由骨髓而來;然而在胰臟組織的生成中還包含了β細胞本身再生作用。本實驗嘗試以異體移殖胰島細胞(islet)治療糖尿病大白鼠,結果發現β細胞複製與分化,且血糖有明顯下降的現象。雖然本實驗對於β細胞分化與生成的作用過程,目前仍無法完全明瞭。無論如何,上述療法的建構,將促使對調控β細胞及幹細胞增殖機制之胰臟內分泌學有更重要的了解。
Human and animal diabetes mellitus were controlled by a dietary treatment supplemented with either a sulfonylurea drug or insulin injection. Insulin injections were inconvenient and the hypoglycemia induced by insulin-overdose could be fatal. Sulfonylurea drugs were administered orally, however, did not typically provide satisfactory control of blood glucose as a starting treatment in 25-30% patients. Therefore, it was imperative to develop a method for the control of human and animal type 1 diabetes mellitus. Recently, insulin gene transferred and expressed in non-pancreatic cells as a means for the treatment of diabetes was developed rapidly in the expanding gene therapy. Retrovirus, lentivirus, adenovirus, adenoassociated virus and herpes simplex had been used as viral vectors, and the constructed viral-insulin gene was successfully transferred into diabetic rat cells. A gene, containing promoter, enhancer and rat type I insulin gene (a-chain, b-chain and signal peptide), was constructed into a retrovirus vector in the study. The constructed viral-insulin gene was transferred into mouse fibroblast cell. The insulin concentration in 3-day cultured mouse fibroblast cells was 4806.35 � 53.72 pg/ml. The insulin concentration for the viral vector containing enhancer and promoter of rat insulin gene was higher than the vector containing only insulin gene by a 61% increase in the cultured mouse fibroblast cells. The enhancer and promoter activity of rat insulin gene would be an important determinant for the expression of insulin gene. The secreted amount of insulin by retrovirus vector contained enhancer/promoter gene in this study could achieve as high concentrations (4806.35 � 53.72 pg/ml) as the insulin injection therapy. Blood glucose decreased significantly for at last 10 days demonstrated that transfection, direction injection of viral-insulin gene into pancreas of diabetic rat, was successful. These studies suggested that the retrovirus vector might be used to transfer the insulin gene in vitro and in vivo. Replacement or regeneration of beta cells in diabetes patients were therapeutic interventions that hold promise in the treatment of diabetes mellitus in the future. It was recognized that these adult pancreatic stem or progenitor beta cells resided in the epithelium of pancreatic ducts, islets, and bone marrow. In addition, it had also been suggested that new islet was also formed by the neogenesis of beta cell residues in existing islets. At present, allogeneic islet transplantation has been explored as a treatment for diabetic rat. After transplantation, the entire mass of beta cells in rats decreased slowly, and subsequently replenished with differentiation beta cells, as well as, blood glucose decreased significantly. Although the mechanism of replacement or regeneration of beta cell has not been fully understand, the development of such therapy will promote to fundamentally understand the mechanisms in regulating beta and stem cells proliferation in the pancreatic endocrinology.
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