9-順式視網酸對粒線體生合成影響之研究

The effects of 9-cis retinoic acid on mitochondrial biogenesis

吳曉梅(Mary Hsiao-Mei Wu)

指導教授 ： 謝榮鴻 博士

臺北醫學大學/公共衛生暨營養學院/保健營養學研究所/碩士(2004年)

摘要

已知粒線體影響生長發育並參予細胞凋亡過程，而對於粒線體生合成控制路徑的瞭解將有助於治療因為粒線體缺陷及匱乏所造成的退化性疾病。本實驗之目的主要建立一個粒線體生合成的細胞模式，探討及篩選影響粒線體生合成的營養素及其調控機轉。本實驗以濃度為0.25 ~ 25 μM的 9-順式視網酸（9-cis retinoic acid, 9-cis RA）培養人類骨癌細胞株143B TK－細胞（human osteosacroma 143B TK－cells）24、48及72小時。之後，以MTS試劑偵測視網酸對細胞存活率之影響，並使用光學顯微鏡觀察細胞形態之變化。利用RT-PCR及西方墨點法分別分析細胞中粒線體mRNA及蛋白質的表現量以評估粒線體生合成之狀況。結果發現，細胞存活率受9-cis RA處理濃度與時數之不同而改變。例如：經25 μM的9-cis RA處理24小時，細胞存活率顯著高於控制組。9-cis RA不改變143B TK－細胞的細胞型態。 9-cis RA顯著地增加粒線體DNA轉錄的氧化磷酸化蛋白質cytochrome c oxidase subunit I （COX I）、 cytochrome c oxidase subunit II （COX II）、 cytochrome c oxidase subunit III （COX III）、NADH dehydrogenase subunit 1（ND I）及ATP synthase subunit 6（ATPase 6）基因的mRNA表現量。例如：經9-cis RA處理24小時後，COX II的mRNA表現量在溶劑組、0.25 μM組、0.5 μM組、1 μM組、2 μM組、5 μM組、10 μM組及25 μM組分別為控制組的0.97 ± 0.09、1.78 ± 0.07、1.67 ± 0.09、1.68 ± 0.24、2.63 ± 0.30、2.05 ± 0.34、2.53 ± 0.39及2.76 ± 0.24倍。同時，9-cis RA增加細胞核DNA轉譯的氧化磷酸化蛋白質NADH-ubiquinone oxidoreductase subunit 9（NDUFA 9）及succinate-ubiquinone oxidoreductase （SDHA）基因的mRNA表現量。另外，retinoid X receptor α（RXRα）及mitochondrial transcription factor A （mtTFA）基因的mRNA表現量亦隨著9-cis RA濃度的增加而顯著地增加。至於在蛋白質表現量部分，9-cis RA增加粒線體DNA轉譯的氧化磷酸化蛋白質COX I、COX II、COX III及細胞核DNA轉譯的氧化磷酸化蛋白質NDUFA9及SDHA 的表現量。例如：經9-cis RA處理72小時後，COX III的蛋白質表現量在溶劑組、0.25 μM組、0.5μM組、1 μM組、2 μM組、5 μM組、10 μM組及25 μM組分別為控制組的0.81 ± 0.20、1.73 ± 0.50、1.71 ± 0.40、1.88 ± 0.64、1.95 ± 0.51、1.96 ± 0.56、1.71 ± 0.53及1.65 ± 0.42倍。然而，9-cis RA在高濃度下卻降低了RXRα蛋白質的表現量。實驗結果顯示，9-cis RA可能藉由活化細胞核接受器RXRα以及mtTFA的大量表現，直接或間接地調控粒線體之生合成。

關鍵字

粒線體生合成 ； 9-順式視網酸 ； 視網酸接受器

並列摘要

In this study, the mRNA and protein levels of mitochondrial DNA-encoded and nuclear DNA-encoded oxidative phosphorylation system subunits were determined by RT-PCR and Western blotting analysis to evaluate the effects of 9-cis retinoic acid (9-cis RA) on mitochondrial biogenesis. Various amounts of 9-cis RA (0.25~25 μM) were given to culture human osteosarcoma 143B TK－ cells for 24, 48, and 72 hours. The MTS assay data showed that the concentration and the treatment time of 9-cis RA would affect the cell survival rate. For examples, after giving 9-cis RA treatments for 24 hours, the cell survival rate in the 25 μM group was significantly higher than in the control group (p < 0.05). However, the cell morphology did not change after 72 hours of treatment. From RT-PCR analysis, the levels of mRNA expression of mitochondrial DNA-encoded cytochrome c oxidase subunit I (COX I), cytochrome c oxidase subunit II (COX II), cytochrome c oxidase subunit III (COX III), NADH dehydro- genase subunit 1(ND1), and ATP synthase subunit 6 (ATPase 6) were significantly increased by 9-cis RA. For instances, after giving 9-cis RA treatments for 24 hours, the mRNA levels of COX II in the vehicle, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 25 μM groups were 0.97 ± 0.09, 1.78 ± 0.07, 1.67 ± 0.09, 1.68 ± 0.24, 2.63 ± 0.30, 2.05 ± 0.34, 2.53 ± 0.39 and 2.76 ± 0.24 folds of the control group. Also, the levels of mRNA expression of nuclear DNA-encoded NADH-ubiquinone oxidoreductase subunit 9 (NDUFA 9) and succinate-ubiquinone oxidoreductase (SDHA) were increased by 9-cis RA. As the concentration of 9-cis RA increased, the levels of mRNA expression of the nuclear DNA-encoded retinoid X receptor α (RXRα) and mitochondrial transcription factor A (mtTFA) were significantly increased. Western blot data revealed that the levels of protein expression of mitochondrial DNA-encoded COX I, COX II and COX III , and nuclear DNA-encoded NDUFA 9 and SDHA were increased by 9-cis RA. For instances, after giving 9-cis RA treatments for 72 hours, the levels of protein expression of COX IIIin the vehicle, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 10 μM and 25 μM groups were 0.81 ± 0.20, 1.73 ± 0.50, 1.71 ± 0.40, 1.88 ± 0.64, 1.95 ± 0.51, 1.96 ± 0.56, 1.71 ± 0.53 and 1.65 ± 0.42 folds of the control group. However, the levels of protein expression of the nuclear receptor, RXRα, were decreased as the concentration of 9-cis RA increased. These results provided evidence of elevation effects of retinoic acid on mitochondrial biogenesis. It is suggested that 9-cis RA may induce mitochondrial biogenesis through activating RXRα and increasing the transcription of mtTFA .

並列關鍵字

mitochondrial biogenesis ； 9-cis retinoic acid ； retinoid X receptor α

參考文獻

Casas, F., Rochard, P., Rodier, A., Cassar-Malek, I., Marchal-Victorion, S., Wiesner, R.J., Cabello, G. & Wrutniak, C. (1999) A variant form of the nuclear triiodothyronine receptor c-ErbAα1 plays a direct role in regulation of mitochondrial RNA synthesis. Mol. Cell. Biol. 19: 7913-7924.

Casas, F., Pineau, T., Rochard, P., Rodier, A., Daury, L., Dauça, M., Cabello, G. & Wrutniak-Cabello, C. (2000b) New molecular aspects of regulation of mitochondrial activity by fenofibrate and fasting. FEBS Letters 482: 71-74.

Casas, F., Dauca, L., Grandemange, S., Busson, M., Seyer, P., Hatier, R., Carazp, A., Cabello, G. ＆ Wrutniak-Cabello, C. (2003) Endocrine regulation of mitochondrial activity: involvement of truncated RXRα and c-Erb Aα1 proteins. FASEB J. 17: 426-436.

Chou, S. Y., Hannah, S. S., Lowe, K. E., Norman, A. W. ＆ Henry, H. L. (1995) Tissue-specific regulation by vitamin D status of nuclear and mitochondrial gene expression in kidney and intestine. Endocrinology 136: 5520-5526.

Clayton, D.A. (1991) Replication and transcription of vertebrate mitochondrial DNA. Annu. Rev. Cell Biol. 7: 453-478.