建立神經毒物誘導及α-Synuclein聚集的帕金森氏症細胞模式做為藥物檢測平台
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2014
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帕金森氏症為中老年人第二大常見的神經退化性疾病，其病理特徵包括靜止性的震顫、僵直、無法運動以及姿勢不穩，導致病發的原因與黑質緻密部的多巴胺神經元損失有關。環境及基因的因素都可能造成帕金森氏症。基因造成帕金森氏症發病機制中，α-synuclein的點突變(例如A30P、A53T 和 E46K)會增加不正常蛋白堆積，Parkin、DJ-1以及PINK1突變會使粒線體功能受損。此外，環境因素以及老化皆會加劇病情惡化。目前沒有有效的藥物可以治療或抑制帕金森氏症的病程。本研究主要使用人類神經母細胞瘤SH-SY5Y細胞，以维生素A酸(retinoic acid)伴隨著巴豆醇-12-十四烷酸酯-13-乙酸酯(12-O-tetradecanoyl-phorbol-13-acetate)進行誘導分化成多巴胺神經元，七天後發現細胞中酪胺酸烴酶(tyrosine hydroxylase，多巴胺神經元的指標；簡稱TH)的表現量增加，並觀察到成熟的型態、較長的神經突觸以及較大的細胞本體。此外，1-methyl-4-phenylpyridinium (MPP+)為粒線體中電子傳遞鏈complex I的抑制劑，使SH-SY5Y細胞死亡，因此利用MPP+ 病理的神經細胞模式，藉測定細胞存活情形來篩選工研院提供植物萃取液有具保護性的植物藥。以細胞存活測試結果發現，前處理NTNU-043、-057、-059、-125、-293、-313、-331、-385、-450、-514，使未分化SH-SY5Y細胞對MPP+引起的細胞毒性有保護性；前處理NTNU-092、-313，則使分化SH-SY5Y細胞對MPP+引起的細胞毒性有保護性。
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting people in their middle and old age. It is characterized by resting tremor, rigidity, akinesia and postural instability and associated with selected loss of dopaminergic (DA) neuron in the substantianigra pars compacta. Combinations of environmental and genetic factors are thought to cause PD. Among the genetic factors contributing to the pathogenesis of PD, mutations in the α-synuclein gene such as A30P, A53T and E46K increased aggregate formation and mutations in Parkin, DJ-1 and PINK1 impaired mitochondrial function. In addition, environmental factors as well as aging are also thought to contribute the development of the disease. No effective treatment for the disease currently urges the development of new agents that may halt the degeneration of PD. In this study, human neuroblastoma SH-SY5Y cells were differentiated toward the DA phenotype in the presence of retinoic acid (RA) follow by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Greatly up-regulated tyrosine hydroxylase (TH, DA neuronal marker) in SH-SY5Y cells was observed following 7 days of differentiation. These TH-positive cells exhibited a mature morphology with long, branched cell processes and large cell bodies. Addition of MPP+ (1-methyl-4-phenylpyridinium), a potent complex I inhibitor in DA neurons, to the above SH-SY5Y cells caused significant cell death compared with untreated cells. This MPP+-induced cell death was used to screen herbal extracts provided by Industrial Technology Research Institute (ITRI). Cell viability assay indicated that pretreatment of herbal extracts NTNU-043, -057, -059, -125, -293, -313, -331, -385, -450, and -514 protected undifferentiated SH-SY5Y cells against MPP+-induced toxicity, whereas pretreatment of herbal extracts NTNU-092 and -313 protected differentiated SH-SY5Y cells against MPP+-induced toxicity.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting people in their middle and old age. It is characterized by resting tremor, rigidity, akinesia and postural instability and associated with selected loss of dopaminergic (DA) neuron in the substantianigra pars compacta. Combinations of environmental and genetic factors are thought to cause PD. Among the genetic factors contributing to the pathogenesis of PD, mutations in the α-synuclein gene such as A30P, A53T and E46K increased aggregate formation and mutations in Parkin, DJ-1 and PINK1 impaired mitochondrial function. In addition, environmental factors as well as aging are also thought to contribute the development of the disease. No effective treatment for the disease currently urges the development of new agents that may halt the degeneration of PD. In this study, human neuroblastoma SH-SY5Y cells were differentiated toward the DA phenotype in the presence of retinoic acid (RA) follow by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Greatly up-regulated tyrosine hydroxylase (TH, DA neuronal marker) in SH-SY5Y cells was observed following 7 days of differentiation. These TH-positive cells exhibited a mature morphology with long, branched cell processes and large cell bodies. Addition of MPP+ (1-methyl-4-phenylpyridinium), a potent complex I inhibitor in DA neurons, to the above SH-SY5Y cells caused significant cell death compared with untreated cells. This MPP+-induced cell death was used to screen herbal extracts provided by Industrial Technology Research Institute (ITRI). Cell viability assay indicated that pretreatment of herbal extracts NTNU-043, -057, -059, -125, -293, -313, -331, -385, -450, and -514 protected undifferentiated SH-SY5Y cells against MPP+-induced toxicity, whereas pretreatment of herbal extracts NTNU-092 and -313 protected differentiated SH-SY5Y cells against MPP+-induced toxicity.
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帕金森氏症, 多巴胺神經元, MPP+, 植物萃取液, Parkinson’s disease, dopaminergic neuron, MPP+, herbal extracts