臺灣博碩士論文加值系統

帕金森病（Parkinson’s disease）是一種影響全球數百萬人的神經退化性疾病。僅次於阿茲海默症，目前僅能依靠藥物治療可以有效減輕症狀，但通常會導致不良的副作用。最近使用細胞替代或間接有益的分泌蛋白組的幹細胞療法已經成為未來治療的希望。儘管已經有各種類型的幹細胞可選擇，但脂肪幹細胞（Adipose-derived stem cell），因其容易獲得、更豐富、道德爭議較少，且能夠分化成多個細胞譜系，而更具優勢。然而，已知使用成體幹細胞治療帕金森氏症比神經元或胚胎幹細胞移植效率低，因此急需改善治療。二甲雙胍(Metformin)具有許多療效，如前驅糖尿病，多囊卵巢綜合徵和妊娠糖尿病。在之前的研究中，二甲雙胍治療患者的癡呆發病率下降至0.67倍。在本研究中，我們評估了二甲雙胍治療後ADSC的治療效果。首先，我們使用MTT測定來證明二甲雙胍0.0625,0.125,0.25,0.5,1和2mM處理後的細胞存活率。結果顯示在2mM下有顯著細胞致死的能力。此外，我們還檢測了二甲雙胍治療後神經生長因子（BDNF和BRN4）和免疫因子（IL6和IL8）基因表達水平。我們發現在二甲雙胍0.25mM和0.125mM處理後BDNF和BRN4基因表達水平顯著上升。 IL6和IL8在二甲雙胍處理後通過使用qPCR測定未顯著上升。這些結果表明二甲雙胍是ADSC預處理的潛在候選者，並且用於評估PD小鼠模型中的治療效果。我們使用神經元行為測試，例如平衡木，旋轉桿和運動活動試驗。將ADSC與MET預處理ADSC移植至紋狀體中，結果表明，在平衡木的行動時間以及腳滑次數有顯著的改善。另外比起ADSC組，MET組在滾輪跑步時間以及於無干擾環境下垂直活動能力皆有顯著提升。我們的研究結果表示ADSC具有改善小鼠運動能力功效，而MET可誘導並增強ADSC的治療能力。因此，我們的研究成果給未來改善帕金森氏症幹細胞治療新的方法。

Parkinson’s disease (PD) is a neurodegenerative disease that affects millions of people worldwide. Drug treatments can effectively reduce symptoms, but often cause unwanted side effects. Stem cell therapies using cells replacement or indirect beneficial secretomes have recently emerged as potential therapeutic strategies. Although various types of stem cells have been proposed as possible candidates, adipose-derived stem cells (ADSCs) are easily obtainable, more abundant, less ethically disputed, and able to differentiate into multiple cell lineages. However, treatment of PD using adult stem cells is known to be less efficacious than neuron or embryonic stem cell transplantation. Therefore, improved therapies are urgently needed. Metformin has many efficacies such us prediabetes, polycystic ovary syndrome and Gestational diabetes. In previous study, the dementia incidence decreased to 0.67-fold in the patients with metformin treatment. In present study, we evaluated the therapeutic effects of ADSC after Metformin treatment. First, we used MTT assay to demonstrate the cell viability after Metformin 0.0625, 0.125, 0.25, 0.5, 1 and 2mM treatment. The results showed significantly decreased cell survival rate in 2mM. In addition, we also detected the nerve growth factors (BDNF and BRN4) and immune factors (IL6 and IL8) gene expression levels after Metformin treatment. We found BDNF and BRN4 gene expression levels significantly upregulated after Metformin 0.25mM and 0.125mM treatment. IL6 and IL8, didn’t significantly upregulate after Metformin treatment by using qPCR assay. These results indicated that Metformin was a potential candidate for ADSC pretreatment and used to evaluate the therapeutic effect in mouse model of PD. We used behavior tests to evaluate the therapeutic effect, such as beam walking, rotarod, and locomotor activity. ADSCs with or without MET pretreatment were transplanted into the striatum. Our findings demonstrated that statistically significant differences between ADSC and MET-ADSC mice in beam walking and foot fault score. MET-ADSC mice showed motor function improve in rotarod and vertical activity compared with ADSC mice. Our findings demonstrated that ADSC transplantation improved motor abilities with varied efficacies and that MET stimulation improved the therapeutic effects. Thus, our results provided the important new strategies to improve stem cell therapies for neurodegenerative diseases in the future.

中文摘要-----------------i
英文摘要-----------------ii
目錄---------------------iv
圖目錄-------------------vii
表目錄-------------------viii
壹、緒論-----------------1
1-1帕金森氏症-----------------1
1-2多巴胺-----------------1
1-3 alpha-synuclein-----------------2
1-4神經幹細胞(Neural stem cell, NSC)-----------------2
1-5 脂肪幹細胞(Adipose derived stem cells, ADSC)-----------------3
1-6二甲雙胍(Metformin, MET)-----------------3
1-7 1-methy1-4-pheny1--phenylpyridinium(MPP+)-----------------3
貳、研究動機與架構-----------------4
參、材料與方法-----------------6
3-1 實驗材料-----------------6
3-1-1實驗細胞-----------------6
3-1-2-1 ADSC medium之配置-----------------6
3-1-2-2 Metformin-----------------7
3-1-2-3 MTT的配置-----------------7
3-1-2-4 DEPC‧H2O的配置-----------------7
3-1-2-5 SYBR Mix混合液-----------------7
3-1-2-6 4%PFA-----------------7
3-1-2-7 20%Sucrose-----------------8
3-1-2-8 30%Sucrose-----------------8
3-1-3 抗體-----------------8
3-1-4實驗儀器與耗材-----------------9
3-2 實驗方法-----------------9
3-2-1 RNA萃取與cDNA備製-----------------9
3-2-2 即時聚合酶連鎖反應(Real-time PCR)-----------------10
3-2-3 ADSC移植-----------------10
3-2-4 動物行為模式分析-----------------10
3-2-4-1 Rotarod -----------------10
3-2-4-2 Locomotor-----------------11
3-2-4-3 Beam Walking-----------------11
3-2-5 冷凍切片-----------------11

肆、結果-----------------12
4-1 ADSC活力測定-----------------12
4-2 MET預處理增強ADSC神經分化因子表現-----------------13
4-3利用行為模式評估帕金森氏症小鼠損傷程度以及修復程度-----------------14
伍、討論-----------------17
陸、參考文獻-----------------18

1.Parkinson, J.,essay on the shaking palsy.(1817).J Neuropsychiatry Clin Neurosci,2002.14(2):p 223-236
2.Barbosa, E.R., J.C. Limongi, and J.L. Cumings, Parkinson’s disease. Psychiatr Clin North Am, 1997.20(4):p 769-790
3.Mizuno, Y., N. Hattori, and h. Matsumine, Neurochemical and neurogenetic correlates of Parkinson''s disease. J Nerochem, 1998.71(3):p. 893-902
4.Hornykiewicz, O., Dopamine and extrapyramidal motor function and dysfunction. Res Publ Assoc Res Nerv Ment Dis, 1972.50:p.390-415
5.Luk, K.C., et al., Pathological alpha-synuclein transmission initiatesv Parkinson-like neurodegeneration in nontransgenic mice. Science, 2012. 338(6109):p.949-953
6.Groenewegen, H.J., The basal ganglia and motor control. Neural plast, 2003. 10(1-2):p107-120
7.Schultz, W., L. Tremblay, and J.R. Hollerman, Changes in behavior-related neuronal activity in the striatum during learning. Trends Neurosci, 2003. 26(6): p. 321-328.
8.Iwai, A., et al., The precursor protein of non-A beta component of Alzheimer''s disease amyloid is a presynaptic protein of the central nervous syistem. Neuron, 1995. 14(2): p. 467-475.
9.Ueda, K., et al., Molecular cloning of cDNA encoding an anrecognized component of amyloid in Altheimer disease. Proc Natl Acad Sci U S A, 1993. 90(23): p. 11282-11286.
10.Cooper, A.A., et al., Alpha-synuclein blocks ER-Golgi trafle andRabl rescues neuron loss in Parkinson''s models. Science, 2006.313(5785): p. 324-328.
11.Perez, R.G., et al., A role for alpha synuclein in the regulation afdopamine biosynthesis. J Neurosci, 2002. 22(8): p. 3090-3099.
12.Sidhu, A., C. Wersinger, and P. Vernier, alpha- Synuclein regulationof the dopaminergic transporter: a possible role in thepathogenesis of Parkinson''s disease. FEBS Lett, 2004. 565(1-3): p.1-5
13.Polymeropoulós, M.I., et al, Mapping of a gene for Parkinson''s disease to chromosome 4421-923. Scienee, 1996. 274(5290): p.1197-1199.
14.Polymeropoulos, M.H., et al., Mutation in the alpha-synuclein geneidentified in families with Parkinson''s disease. Science, 1997276(5321): p. 2045-2047
15.Gallegos, S., et al., Features of alpha-synuclein that could explain the progression and irreversibility of Parkinson''s disease. FrontNeurosci, 2015. 9: p. 59.
16.Reynolds, B.A and S. Weiss, Generation of heurons and astrocytes from isolaed cells of the adull mammalian central nervous system. Science, 1992, 255(5052): p.1707-1710.
17.Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Science 1999;284(5411):143-147.
18.Reid AJ, Sun M, Wiberg M, Downes S, Terenghi G, Kingham PJ. Nerve repairwith adipose-derived stem cells protects dorsal root ganglia neurons from apoptosis. Neuroscience 2011;199:515-22.
19.Gimble JM, Katz AJ, Bunnell BA. Adipose-Derived Stem Cells for RegenerativeMedicine. Circulation Research 2007;100(9):1249-1260.
20.Trzaska KA, Rameshwar P. Dopaminergic Neuronal Differentiation Protocol for Human Mesenchymal Stem Cells. In: Vemuri M, Chase LG, Rao MS, editors. Mesenchymal Stem Cell Assays and Applications. Totowa, NJ: Humana Press; 2011. p 295-303.
21.Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human Adipose Tissue Is a Source of Multipotent Stem Cells. Molecular Biology of the Cell 2002;13(12):4279-4295.
22.Anghileri E, Marconi S, Pignatelli A, Cifelli P, Galié M, Sbarbati A, Krampera M, Belluzzi O, Bonetti B. Neuronal Differentiation Potential of Human Adipose-Derived Mesenchymal Stem Cells. Stem Cells and Development 2008;17(5):909-916.
23.Bruce A.Bunnell ., et al. Adipose-derived stem cells: Isolation, expansion and differentiation. Methods Volume 45, Issue 2, June 2008, Pages 115-120
24.Marchetti P, Benzi L, Cecchetti P et al. Plasma biguanide levels are correlated with metabolic effects in diabetic patients. Clin Pharmacol Ther. 1987; 41:450-454.
25.Rosina Pryor., et al. Repurposing metformin: an old drug with new tricks in its binding pockets. Biochem J. 2015 Nov 1; 471(Pt 3): 307–322.
26.Nicklas, W.J., I. Vyas, and R.E. Heikkila, Inhibition of NADIH-linked oidation brain mitochondria by I-methyl-4-phenyl-pyridine, a metabolite of the neurotoxin, I-methyl-4phenyl-1,25,6-lerahyahopyridime Life.Sci, 1985 36(26): p. 2503-2508.
27.Schapira, .?B., et al., Mitochondrial complex I deficiency inParkinson''s disease. Lancet, 1989. 1(8649):p. 1269.
28.Acuna-Castroviejo, D, et al., Melatonin is protective against MPTP-induced strtatal and hippocampal lesions. Life Sci, 1997 60(2): p. PL23-9
29.Dexter, D.T., et al., Increased nigral iron content and alterations in other metal ions occurring in brain in Parkinson''s disease. J Neurochem, 1989. 52(6): p. 1830-1836.
30.Poirier, J. and A. Barbeau, A catalyst fiunction for MPTP in superoxide formation. Biochem Biophys Res Commun, 1985, 131(3): p. 1284-9.
31.Rossetti, Z.L., et al., I-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and free radicals in vitro. Biochem Pharmacol, 1988.37(23):p.4573-4
32.Kefalopoulou Z., et al. Long-term Clinical Outcome of Fetal Cell Transplantation for Parkinson Disease Two Case Reports. JAMA neurology 2014;71(1):83-87.
33.Kawasaki H., et al. Induction of Midbrain Dopaminergic Neurons from ES Cells by Stromal Cell–Derived Inducing Activity. Neuron 2000;28(1):31-40.
34.Masgutov RF., et al. Human adipose-derived stem cells stimulate neuroregeneration. Clinical and Experimental Medicine 2016;16(3):451-461.
35.Schwerk A., et al. Adipose-derived human mesenchymal stem cells induce long-term neurogenic and anti-inflammatory effects and improve cognitive but not motor performance in a rat model of Parkinson''s disease. Regenerative Medicine 2015;10(4):431-446.