臺灣博碩士論文加值系統

視網酸X受體(RXR-α)是核接受器超級家族的其中ㄧ員，會與其它的接受器，如peroxisome proliferator activated receptor (PPAR), retinoic acid receptor (RAR), thyroid receptor, and vitamin D receptor (VDR)等形成異質雙體而結合到基因上進行轉錄調節。2003年Casas等學者研究發現在粒線體基質中發現一個44 kDa截短形式的RXR-α (mtRXR-α)；本實驗利用RXR-α表現載體(RXR-α expression vectors)為了要更明顯的觀察RXR-α進入粒線體的現象。目前9-cis retinoic acid已被鑑定為RXR之配體(ligand)，有許多研究指出retinoids可以抑制細胞的增生及促進細胞的分化；A23187為一種鈣離子載體(calcium ionophore)，在先前的研究發現可促進細胞內鈣離子增加，當細胞內鈣離子增加就會誘導鈣激蛋白酶(calpain)活化，在1996年Nishiwaki等學者發現calpain可以將RXR-α截成44kDa。因此，本實驗之目的為探討RXR-α蛋白質過度表現(RXR-α overexpression)後對於粒線體生合成的影響為何。首先利用細胞內生性的calpain將RXR-α截短後送入粒線體，投予A23187發現會增加細胞內的鈣離子以及calpain活性。利用Real-Time PCR及西方點墨法分別分析細胞中粒線體mRNA和蛋白質的表現量以評估粒線體生合成之狀況，結果顯示轉染RXR-α再投予A23187以及9-cis retinoic acid並不影響細胞轉譯及轉錄作用。以共軛焦顯微鏡觀察RXR-α進入粒線體的情形，結果發現RXR-α過量表現後投予A23187以及投予1 μM, 5 μM 以及25 μM的9-cis retinoic acid可使RXR-α進入粒線體。使用流式分析儀分析粒線體膜電位(ΔΨ)和細胞凋亡程度，結果顯示A23187也會促進粒線體膜電位滲漏及細胞凋亡的程度。因此由本實驗推論RXR-α並不參與細胞粒線體轉譯及轉錄作用，但投予9-cis retinoic acid在氧化磷酸化系統上(OXPHOS)的蛋白質都有降低的趨勢，由此推論當細胞內RXR-α過量表現(overexpression)時，9-cis retinoic acid可能會促進配體活化反應(ligand-activation)，這個機制在未來的研究也將深入探討。

The retinoid X receptor alpha (RXR-α) is one of the nuclear receptor superfamily members that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator activated receptor (PPAR), retinoic acid receptor (RAR), thyroid receptor, and vitamin D receptor (VDR). In 2003, Casas discovered truncated RXR-α in mitochondria and called mtRXR-α. 9-cis retinoic acid has been identified to be the ligand of RXR and more studies have shown that retinoids inhibit cell proliferation but increase cell differentiation. A23187, a calcium ionophore, be discovered could increase cellular calcium concentration and induce calpain activation. In order to study the mechanism of RXR-α translocation and regulation of mitochondrial biogenesis and to investigate the effects of mitochondrial biogenesis on neuron degeneration, we created a cellular studied model with RXR-α overexpression on U87-MG astrocytoma cells followed by A23187 treatment. The results showed RXR-α protein expression 2.5 folds higher than control when RXR-α overexpression in U87-MG cells. The levels of mRNA and protein of respiratory enzyme subunits from both of mtDNA-encoded genes and nuclear DNA-encoded genes were determined by real-time PCR and western blot, respectively. There were no differences of transcription and translation levels of cells giving A23187 and 9-cis retinoic acid after RXR-α overexpression. However, cells with RXR-α overexpression followed by 1 μM and 5 μM 9-cis retinoic acid treatment showed lower mtDNA encoded protein ND6 compare to control. The proportions are 0.798±0.239 and 0.42±0.141 when compared to control, respectively. The leves of RXR-α translocation into mitochondria in cells given A23187 and 1 μM, 5 μM and 25 μM 9-cis retinoic acid after transfect RXR-α were observed by confocal microscope. The mitochondrial membrane protential and apoptosis level analyzed by flow cytometry and showed A23187 treatment could induce mitochondrial membrane protential loss. The ratios are 21.07±1.544, 6.81±0.327, 9.53±0.036 in the control, in the Mock+A23187, and RXR-α +A23187 groups, respectively. The A23187 also induced cell apoptosis, cell early apoptosis ratios are 3.16±0.28%, 15.66±4.86%, 11.49±0.16%, 12.17±0.92% in control, control+A23187, Mock+A23187 and RXR-α+A23187 groups, respectivtly. In this study, results indicated that RXR-α may not participate in mitochondrial transcription and translation. However, given 9-cis retinoic acid could decreased the protein levels of oxidative phosphorylation system including complex V alpha, COX I and the COX II. These studies suggested that cellular RXR-α overexpression followed given 9-cis retinoic acid could induce ligand-activation where RXR-α translocated into mitochondria. The same results are determined in cells incubated with A23187.

中文摘要 I
Abstract III
目錄 V
圖目錄 VIII
表目錄 X
第一章 研究動機 1
第二章 文獻回顧 2
第一節 人類神經膠細胞(Glioma cell)的概述 2
一、 人類神經膠細胞的誕生及生理功能 2
二、 人類神經膠細胞的分級 2
三、鈣離子載體(Ionophore)活化鈣激活蛋白酶(calpains) 3
第二節 粒線體基因 4
第三節 RXR-α 基因和功能 5
第四節 RXR-α 調節細胞核基因表現之機制 6
第五節 RXR-α 調節粒線體基因表現之機制 6
第六節 粒線體於星狀細胞中的功能 7
第七節 視網酸與神經膠細胞的調節作用 9
第三章 研究架構 11
第四章 實驗材料與方法 14
第一節 細胞培養 14
ㄧ、細胞株 14
二、培養液配製 14
三. RXR-α過量表現質體 15
第二節 勝任細胞 (competent cells) 之製備 15
一、轉型作用 (transformation) 15
二、小量質體DNA之萃取 15
三、大量質體DNA之萃取 16
四、DNA定量 17
五、空載體製備 18
第三節 人類RXR-α基因之轉殖(transfection) 18
第四節 細胞內鈣離子表現量 19
第五節 Calpain 活性分析 19
第六節 粒線體蛋白質萃取 20
第七節 蛋白質濃度的定量 21
第八節 西方點墨法(Western Blot) 22
第九節 引子設計 23
第十節 反轉錄聚合酶鏈鎖反應(RT-PCR Analysis) 23
第十一節 流式細胞儀(Flow Cytometry) 24
第十二節 共軛焦顯微鏡(Confocal Microscope) 25
第五章 結果 27
第一節 轉染RXR-α 後投予A23187 對於U87-MG細胞表現之影響 27
一、轉染RXR-α表現質體之RXR-α蛋白表現情形 27
二、轉染RXR-α 後投予A23187對於細胞鈣離子及calpain活性之影響27
第二節 轉染RXR-α 後投予A23187 對於U87-MG細胞mRNA和蛋白質影響 28
一、轉染RXR-α對mtDNA所轉錄的氧化磷酸化系統上蛋白質的mRNA表現量的影響 28
二、轉染RXR-α對細胞核DNA所轉錄的氧化磷酸化系統上蛋白質的mRNA表現量之影響 28
三、轉染RXR-α對粒線體轉錄因子A的mRNA和蛋白質表現量之影響29
第三節 轉染RXR-α 後投予A23187會使RXR-α轉移進入粒線體 30
第四節 轉染RXR-α 後投予A23187對於U87-MG細胞的影響 30
一、轉染RXR-α 及投予A23187對U87-MG細胞粒線體膜電位差的影響30
二、投予A23187促進U87-MG細胞死亡 31
第五節 轉染RXR-α 後投予9-cis retinoic acid對U87-MG細胞的蛋白質表現量影響 31
一、轉染RXR-α 後投予9-cis retinoic acid對細胞核DNA所轉錄的氧化磷酸化系統上蛋白質表現量並無影響 32
二、 轉染RXR-α 後投予9-cis retinoic acid對粒線體DNA所轉錄的氧化磷酸化系統上蛋白質表現量的影響 33
第六節 轉染RXR-α 後投予9-cis retinoic acid對RXR-α轉移進入粒線體之影響 34
一、 未投予9-cis retinoic acid對RXR-α轉移進入粒線體之影響 34
二、 在投予1 μM 9-cis retinoic acid後，經RXR-α過度表現已發現RXR-α轉移進入粒線體 35
三、 在投予5μM 9-cis retinoic acid後，經已發現RXR-α轉移進入粒線體 36
四、在投予25 μM 9-cis retinoic acid後發現RXR-α轉移進入粒線體 36
第六章 討論 38
第一節 過度表現RXR-α後投予A23187對U87-MG細胞RXR-α進入粒線體表現影響 38
第二節 投予A23187對於細胞內鈣離子和calpian活性表現量影響39
第三節 Calpian活性對於RXR-α轉移進入粒線體的表現 40
第四節 過度表現RXR-α後投予A23187對U87-MG細胞粒線體生合成影響 40
1、過度表現RXR-α後投予A23187對U87-MG細胞mRNA表現 41
2、過度表現RXR-α後投予A23187對U87-MG細胞影響 42
第五節 過度表現RXR-α後投予9-cis retinoic acid對U87-MG細胞粒線體生合成及轉移進入粒線體表現 42
1、過度表現RXR-α後投予9-cis retinoic acid對U87-MG細胞影響43
第六節 過度表現RXR-α後投予9-cis retinoic acid對RXR-α進入粒線體的表現 44
第七章 結論 45
第八章 參考文獻 46
附錄 80

Abril J, de Heredia ML, Gonzalez L, Cleries R, Nadal M, Condom E, Aguilo F, Gomez-Zaera M & Nunes V (2008) Altered expression of 12S/MT-RNR1, MT-CO2/COX2, and MT-ATP6 mitochondrial genes in prostate cancer. Prostate 68, 1086-1096.
Ahmed SM, Rzigalinski BA, Willoughby KA, Sitterding HA & Ellis EF (2000) Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons. J Neurochem 74, 1951-1960.
Andrade FH, McMullen CA & Rumbaut RE (2005) Mitochondria are fast Ca2+ sinks in rat extraocular muscles: a novel regulatory influence on contractile function and metabolism. Invest Ophthalmol Vis Sci 46, 4541-4547.
Bambrick L, Kristian T & Fiskum G (2004) Astrocyte mitochondrial mechanisms of ischemic brain injury and neuroprotection. Neurochem Res 29, 601-608.
Berdanier CD, Everts HB, Hermoyian C & Mathews CE (2001) Role of vitamin A in mitochondrial gene expression. Diabetes Res Clin Pract 54 Suppl 2, S11-27.
Bouterfa H, Picht T, Kess D, Herbold C, Noll E, Black PM, Roosen K & Tonn JC (2000) Retinoids inhibit human glioma cell proliferation and migration in primary cell cultures but not in established cell lines. Neurosurgery 46, 419-430.
Casas F, Daury L, Grandemange S, Busson M, Seyer P, Hatier R, Carazo A, Cabello G & Wrutniak-Cabello C (2003) Endocrine regulation of mitochondrial activity: involvement of truncated RXRalpha and c-Erb Aalpha1 proteins. Faseb J 17, 426-436.
Chattopadhyay N, Butters RR & Brown EM (2001) Agonists of the retinoic acid- and retinoid X-receptors inhibit hepatocyte growth factor secretion and expression in U87 human astrocytoma cells. Brain Res Mol Brain Res 87, 100-108.
Cullingford TE, Bhakoo K, Peuchen S, Dolphin CT, Patel R & Clark JB (1998) Distribution of mRNAs encoding the peroxisome proliferator-activated receptor alpha, beta, and gamma and the retinoid X receptor alpha, beta, and gamma in rat central nervous system. J Neurochem 70, 1366-1375.
Das A, Banik NL & Ray SK (2008) Molecular Mechanisms of the Combination of Retinoid and Interferon-gamma for Inducing Differentiation and Increasing Apoptosis in Human Glioblastoma T98G and U87MG Cells. Neurochem Res.
Dragnev KH, Rigas JR & Dmitrovsky E (2000) The retinoids and cancer prevention mechanisms. Oncologist 5, 361-368.
Everts HB, Claassen DO, Hermoyian CL & Berdanier CD (2002) Nutrient-gene interactions: dietary vitamin A and mitochondrial gene expression. IUBMB Life 53, 295-301.
Gaemers IC, Sonneveld E, van Pelt AM, Schrans BH, Themmen AP, van der Saag PT & de Rooij DG (1998) The effect of 9-cis-retinoic acid on proliferation and differentiation of a spermatogonia and retinoid receptor gene expression in the vitamin A-deficient mouse testis. Endocrinology 139, 4269-4276.
Giffard R & Ouyang Y (2004) Effect of overexpression of protective genes on mitochondrial function of stressed astrocytes. J Bioenerg Biomembr 36, 313-315.
Gonzalez J & Gilbert MR (2005) Treatment of astrocytomas. Curr Opin Neurol 18, 632-638.
Huang G, Chen Y, Lu H & Cao X (2007) Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death. Cell Death Differ 14, 327-337.
Kajitani N, Kobuchi H, Fujita H, Yano H, Fujiwara T, Yasuda T & Utsumi K (2007) Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase. Biosci Biotechnol Biochem 71, 2701-2711.
Karmakar S, Banik NL, Patel SJ & Ray SK (2007) Combination of all-trans retinoic acid and taxol regressed glioblastoma T98G xenografts in nude mice. Apoptosis 12, 2077-2087.
Kleinschmidt-DeMasters BK, Orr EA, Savelieva E, Owens GC & Kruse CA (1999) Paucity of retinoic acid receptor alpha (RAR alpha) nuclear immunostaining in gliomas and inability of retinoic acid to influence neural cell adhesion molecule (NCAM) expression. J Neurooncol 41, 31-42.
Lee HY, Dawson MI, Walsh GL, Nesbitt JC, Eckert RL, Fuchs E, Hong WK, Lotan R & Kurie JM (1996) Retinoic acid receptor- and retinoid X receptor-selective retinoids activate signaling pathways that converge on AP-1 and inhibit squamous differentiation in human bronchial epithelial cells. Cell Growth Differ 7, 997-1004.
Li JJ & Xie D (2007) Cleavage of focal adhesion kinase (FAK) is essential in adipocyte differentiation. Biochem Biophys Res Commun 357, 648-654.
Lin XF, Zhao BX, Chen HZ, Ye XF, Yang CY, Zhou HY, Zhang MQ, Lin SC & Wu Q (2004) RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells. J Cell Sci 117, 5609-5621.
Mangelsdorf DJ & Evans RM (1995) The RXR heterodimers and orphan receptors. Cell 83, 841-850.
Maracchioni A, Totaro A, Angelini DF, Di Penta A, Bernardi G, Carri MT & Achsel T (2007) Mitochondrial damage modulates alternative splicing in neuronal cells: implications for neurodegeneration. J Neurochem 100, 142-153.
Marceau G, Gallot D, Lemery D & Sapin V (2007) Metabolism of retinol during mammalian placental and embryonic development. Vitam Horm 75, 97-115.
Matsushima-Nishiwaki R, Shidoji Y, Nishiwaki S, Moriwaki H & Muto Y (1996) Limited degradation of retinoid X receptor by calpain. Biochem Biophys Res Commun 225, 946-951.
Matyja E (2000) Aluminum enhances glutamate-mediated neurotoxicity in organotypic cultures of rat hippocampus. Folia Neuropathol 38, 47-53.
Rastinejad F (2001) Retinoid X receptor and its partners in the nuclear receptor family. Curr Opin Struct Biol 11, 33-38.
Pfahl M (1993) Signal transduction by retinoid receptors. Skin Pharmacol 6 Suppl 1, 8-16.
Robb SJ, Robb-Gaspers LD, Scaduto RC, Jr., Thomas AP & Connor JR (1999) Influence of calcium and iron on cell death and mitochondrial function in oxidatively stressed astrocytes. J Neurosci Res 55, 674-686.
Ruiz-Lozano P, Smith SM, Perkins G, Kubalak SW, Boss GR, Sucov HM, Evans RM & Chien KR (1998) Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos. Development 125, 533-544.
Sanai N, Alvarez-Buylla A & Berger MS (2005) Neural stem cells and the origin of gliomas. N Engl J Med 353, 811-822.
Sharma AK & Rohrer B (2004) Calcium-induced calpain mediates apoptosis via caspase-3 in a mouse photoreceptor cell line. J Biol Chem 279, 35564-35572.
Takano Y, Adachi S, Okuno M, Muto Y, Yoshioka T, Matsushima-Nishiwaki R, Tsurumi H, Ito K, Friedman SL, Moriwaki H, Kojima S & Okano Y (2004) The RING finger protein, RNF8, interacts with retinoid X receptor alpha and enhances its transcription-stimulating activity. J Biol Chem 279, 18926-18934.
Voloboueva LA, Suh SW, Swanson RA & Giffard RG (2007) Inhibition of mitochondrial function in astrocytes: implications for neuroprotection. J Neurochem 102, 1383-1394.
Yasmin R, Williams RM, Xu M & Noy N (2005) Nuclear import of the retinoid X receptor, the vitamin D receptor, and their mutual heterodimer. J Biol Chem 280, 40152-40160.
Zhang XK (2007) Targeting Nur77 translocation. Expert Opin Ther Targets 11, 69-79.
Zhu H, Gao W, Jiang H, Wu J, Shi YF & Zhang XJ (2007) Calcineurin mediates acetylcholinesterase expression during calcium ionophore A23187-induced HeLa cell apoptosis. Biochim Biophys Acta 1773, 593-602.
Zundorf G, Kahlert S & Reiser G (2007) Gap-junction blocker carbenoxolone differentially enhances NMDA-induced cell death in hippocampal neurons and astrocytes in co-culture. J Neurochem 102, 508-521.