生活在高壓力工作的人們，缺乏足夠運動的生活方式經常引發多種代謝綜合症或者是心血管疾病。研究表明運動不僅可以增加免疫系統，減少民間疾病的發生，還可以幫助患有神經系統疾病的患者恢復體力的喪失。然而，運動對於神經保護或者是神經元分化的有益效果仍不清楚，Irisin是一種新的肌動蛋白，它已被證明是由骨骼肌分泌的纖連蛋白III型結構域的蛋白質5經由蛋白質水解酶切割而來的。研究表明，Irisin可以有效的誘導白色脂肪組織轉化成棕色脂肪組織並且改善第二型糖尿病。此外，Irisin也被作為神經細胞存活和發育的潛在調節劑。因此在這項研究中，我們調查了Irisin和Irisin-mutant重組蛋白在神經分化和神經元保護中的作用。根據晶體結構和生化數據的支持，Irisin可以藉由鹽橋區域和雙硫鍵形成二聚體，因此我們生產出IrisinWT以及將Irisin點突變形成IrisinR75E、IrisinC87A以及IrisinR75E/C87A去完成我們的目標，藉由增殖和遷移的試驗，表明IrisinWT以劑量方式刺激SH-SY5Y細胞的增殖和遷移，另外，IrisinWT也可以減少6-OHDA以及CoCl2所誘導的細胞死亡，在RT-PCR和Western blot的實驗中顯示出IrisinWT在視黃酸(RA)誘導的類神經元細胞中增加mRNA以及蛋白質水平。總之我們的研究結果表明，IrisinWT可以保護神經細胞免於神經毒素6-OHDA所誘導的細胞損傷，也可以對於CoCl2所誘導的細胞在缺氧狀態下具有保護作用。在另一方面，Irisin-突變蛋白只有部分或無法保護神經元細胞經由神經毒素6-OHDA以及CoCl2所誘導的細胞傷害。綜合上述，實驗結果顯示：Irisin二聚體形成對其生物功能是重要的，並且雙硫鍵對於Irisin形成二聚體是關鍵的。

People who live in high-pressure work environment and lacking enough exercise of lifestyle frequently elicit several metabolic syndromes or cardiovascular diseases. Studies have shown that exercise not only enhance immune system and reduce the incidence of civil diseases, but also help patients with neurological disorders to regain lost physical abilities. However, how beneficial effects of exercise on neuroprotection or neuronal differentiation is still unclear. Irisin, a newly myokine protein, which has been shown to be secreted from transmembrane protein fibronectin type III domain containing 5 (FNDC5) of skeletal muscle. Studies have indicated that Irisin is efficient to induce converting differentiation of white adipose tissue into brown adipose tissue and improve type 2 diabetes. Moreover, Irisin is also shed as a potential regulator in neuronal survival and development. Thus, in this study, we investigated the role of IrisinWT and Irisin-mutnat recombinant proteins in neuronal differentiation and neuron protection. According to the crystal structure and biochemical results supported that IrisinWT can form a dimerization by salt bridge region and disulfide bond, we thus generated IrisinWT and point mutation of IrisinR75E, IrisinC87A and IrisinR75EC87A proteins to address this aim. By proliferation and migration assays, our results indicated that IrisinWT stimulate the cell proliferation and migration in SH-SY5Y cells in dose manner. In addition, IrisinWT reduced the cell death induced by 6-OHDA or CoCl2 in SH-SY5Y cells. Moreover, RT-PCR and Western blot results revealed that an elevation of mRNA and protein level of IrisinWT in Retinoic acid (RA)-induced neuron-like cells. In summary, our results suggested that IrisinWT protect neuron cells against neurotoxin 6-OHDA-induced cell damage and protect neuron cells against CoCl2-induced cell damage in a hypoxic environment. On the other hand, Irisin-mutant proteins were only partially or unable to protect neuronal cells from neurotoxin 6-OHDA-induced cell injury and protect neuronal cells from CoCl2-induced cell damage in a hypoxic environment. Taken together, the results implied that the dimerization of Irisin is important for its bio-function, and the formation of disulfide bonds is critical to the dimerization of Irisin.

論文審定書 i
論文公開授權書 ii
中文摘要 iii
英文摘要 iv
INDEX vi
Introduction 1
Specific aims 11
Materials and Methods 12
Results 19
Discussion 27
Figures and legends 31
Appendix 50
References 51

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