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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林君榮 | |
dc.contributor.author | Chen-Chi Hsu | en |
dc.contributor.author | 徐晨綺 | zh_TW |
dc.date.accessioned | 2021-06-15T06:45:48Z | - |
dc.date.available | 2012-10-07 | |
dc.date.copyright | 2011-10-07 | |
dc.date.issued | 2011 | |
dc.date.submitted | 2011-06-24 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/48081 | - |
dc.description.abstract | 肉鹼 (Carnitine)是一種極性很高的化合物,必須利用轉運蛋白的幫助送入細胞中,其主要生理功能在於幫助長鏈脂肪酸 (long-chain fatty acids) 從細胞質轉移至粒線體中進行β氧化反應 (β-oxidation) 以提供細胞所需之能量。胎兒在發育期間對於肉鹼的製造能力不完全,為了維持胎兒肉鹼的需求,在胚胎發育時肉鹼必須從母親透過胎盤供應給胎兒,由於第二新型有機陽離子轉運蛋白 (OCTN2)對於肉鹼的運輸具有很高的親和力,本研究的目的是探討在BeWo細胞中,thiazolidinediones對胎盤OCTN2表現之影響以及其中可能所涉及的機轉,並且在人類胎盤組織中,子癲前症的胎盤組織上的OCTN2表現也一併作探討。
本研究以人類絨毛膜癌細胞BeWo做為研究的模組。實驗過程中利用反轉錄及定量聚合酶鏈鎖反應探討細胞中mRNA表現量的變化,並以西方墨點法來探討細胞中蛋白質表現。結果顯示在給予10 μM troglitazone的條件下,OCTN2的mRNA表現在給藥後72小時後顯著下降約60%,相較之下,給予相同濃度的rosiglitazone或是pioglitazone 72小時之後並沒有看到對OCTN2 mRNA表現的影響。OCTN2蛋白質的表現與mRNA的結果一致。Troglitazone的處理對於調控OCTN2的轉錄因子PPARα以及其執行功能的夥伴RXRα其mRNA的表現有明顯的下降。相反的,rosiglitazone以及pioglitazone並沒有對於PPARα或是RXRα的mRNA造成影響。經由troglitazone的處理,BeWo細胞中HIF-1α mRNA以及蛋白質的表現會增加,同時也造成p38活性增加以及Erk活性下降。除此之外,在低氧環境下的人類胎盤組織中,OCTN2及PPARα的表現皆會明顯下降。而跟缺氧相關的子癲前症 (preeclampsia)其胎盤上的OCTN2及PPARα與正常胎盤組織相比也是明顯的較低。 整體而言,在人類絨毛膜癌細胞BeWo中,troglitazone會影響OCTN2及HIF-1α的表現。這些改變有可能是透過 mitogen-activated protein kinase (MAPK)的途徑所致。除此之外,低氧環境會使人類胎盤組織中OCTN2及PPARα表現的降低,同時在子癲前症的胎盤上,OCTN2以及PPARα的表現相較於正常胎盤也有明顯的減少。 | zh_TW |
dc.description.abstract | Carnitine is important for the transfer of acry-carnitine with long-chain fatty acids, which is then transported into mitochondria for β-oxidation. The fetus cannot synthesize adequate amount of carnitine, and the active transfer of carnitine from the mother to the fetus is important. The novel organic cation transporter 2 (OCTN2) is a high-affinity carnitine transporter. In current study, we investigate the effects of thiazolidinediones on placental OCTN2 expression and the underlying mechanisms in human choriocarcinoma BeWo cells. In addition, OCTN2 expression was measured in human placentas isolated from pregnancy with or without preeclampsia.
After treatment of troglitazone, rosiglitazone and pioglitazone in BeWo cells, the mRNA and protein levels were analyzed by RT-qPCR and Western blotting, respectively. The results showed that, after 72 hr of treatment with troglitazone (10 μM), OCTN2 mRNA expression was significantly reduced by 60% in BeWo cells. In contrast, rosiglitazone (10 μM) and pioglitazone (10 μM) did not cause any change in OCTN2 mRNA expression in BeWo cells. Results of OCTN2 protein expression were consistent with those in mRNA. Troglitazone treatment significantly reduced both PPARα and RXRα mRNA levels. In contrast, treatment with rosiglitazone or pioglitazone did not reduce PPARα or RXRα mRNA expression. Both mRNA and protein levels of HIF-1α were increased by troglitazone treatment in BeWo cells, along with an increase in p38 activity and a decrease in Erk activity. In addition, the expression of OCTN2 and PPARα protein was significantly reduced in human placenta explants by hypoxia treatment and in placentas isolated from preeclampsia. In conclusion, troglitazone, but not rosiglitazone or pioglitazone, regulates OCTN2 and HIF-1α expression in placental choriocarcinoma BeWo cells and this effect may be mediated by mitogen-activated protein kinase. Hypoxia treatment caused a decrease in OCTN2 and PPARα expression in human placenta explants, which resembles the findings in preeclamptic placentas. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T06:45:48Z (GMT). No. of bitstreams: 1 ntu-100-R98423005-1.pdf: 1697450 bytes, checksum: 6e615a69d6a5a4672c628ef8b3d9f0c9 (MD5) Previous issue date: 2011 | en |
dc.description.tableofcontents | Abstract i
中文摘要 iii 目錄 v 圖目錄 x 第1章 文獻回顧 1 1.1. 胎盤及胎盤細胞株 1 1.2. 藥物在胎盤之運輸及影響 2 1.3. 第二型糖尿病藥物Thiazolidinediones (TZDs) 及過氧化體增殖體活化受體 (Peroxisome proliferator-activated receptors,PPARs) 3 1.4. 新型有機陽離子轉運蛋白 (Novel organic cation transporters,OCTNs)及肉鹼 (Carnitine) 7 第2章 研究目的 12 第3章 實驗材料 13 3.1. 細胞培養 13 3.1.1. 試劑 13 3.1.2. 材料與設備 13 3.2. 臨床人類胎盤組織收集 14 3.2.1. 試劑 14 3.2.2. 材料與設備 15 3.3. 藥物與低氧處理 15 3.3.1. 試劑 15 3.3.2. 材料與設備 15 3.4. 反轉錄及定量聚合酶鏈鎖反應(Reverse transcription-quantitative polymerase chain reaction, RT-qPCR) 16 3.4.1. 試劑 16 3.4.2. 材料與設備 17 3.5. 膜蛋白萃取 (Membrane protein extraction) 17 3.5.1. 試劑 17 3.5.2. 材料與設備 17 3.6. 蛋白萃取 (Lysate extraction) 18 3.6.1. 試劑 18 3.6.2. 材料與設備 18 3.7. 核蛋白萃取 (Nuclear protein extraction) 18 3.7.1. 試劑 18 3.7.2. 材料與設備 19 3.8. 蛋白質濃度測定 (Bio-Rad DC protein assay) 19 3.8.1. 材料與設備 19 3.9. 西方墨點法 (Western Blot) 19 3.9.1. 試劑 19 3.9.2. 一級抗體 20 3.9.3. 二級抗體 21 3.9.4. 材料與設備 21 3.10. 報導基因分析 (Reporter Assay) 21 3.10.1. 試劑 21 3.10.2. 材料與設備 22 3.11. 活性氧化物 (Reactive oxygen species, ROS)測定 22 3.11.1. 試劑 22 3.11.2. 材料與設備 22 3.12. 肉鹼在BeWo細胞攝取量的研究 (Uptake study) 22 3.12.1. 試劑 22 3.12.2. 材料與設備 23 3.13. 其他 23 3.13.1. 其他儀器 23 第4章 實驗方法 24 4.1. 細胞培養 24 4.1.1. BeWo細胞株培養 24 4.1.2. HepG2(Human hepatocellular carcinoma cell line)細胞培養 24 4.1.3. HEK293(Human embryonic kidney 293 cells)細胞培養 24 4.2. 臨床人類胎盤組織收集 25 4.3. 藥物與低氧處理 25 4.4. 反轉錄及定量聚合酶鏈鎖反應(Reverse transcription-quantitative polymerase chain reaction, RT-qPCR) 26 4.4.1. RNA抽取 (RNA extraction) 26 4.4.2. RT (Reverse transcription) 26 4.4.3. QPCR (Quantitative polymerase chain reaction) 27 4.5. 膜蛋白萃取 (Membrane protein extraction) 27 4.5.1. 細胞膜蛋白萃取 27 4.5.2. 組織膜蛋白萃取 27 4.6. 蛋白萃取 (Lysate extraction) 28 4.6.1. 細胞蛋白萃取 28 4.6.2. 組織蛋白萃取 28 4.7. 核蛋白萃取 (Nuclear protein extraction) 28 4.8. 蛋白質濃度測定 (Bio-Rad DC protein assay) 29 4.9. 西方墨點法 (Western Blot) 29 4.10 報導基因分析 (Reporter Assay) 30 4.10.1. 細胞轉染 (Transfection) 30 4.10.2. 冷光活性分析 (Luciferase Assay) 31 4.11. 活性氧化物 (Reactive oxygen species, ROS)測定 31 4.12. 肉鹼在BeWo細胞攝取量的研究 (Uptake study) 32 4.13. 數據分析 32 第5章 實驗結果 34 5.1. Thiazolidinediones藥物對於BeWo細胞之OCTN2 mRNA的影響 34 5.2. Thiazolidinediones藥物對於OCTN2 promoter的影響 34 5.3. BeWo細胞在低氧環境下WY14643對於肉鹼攝取的影響 35 5.4. 胎盤組織在低氧環境其OCTN2和PPARα之表現 35 5.5. 子癲前症患者其胎盤組織上其OCTN2和PPARα之表現 35 5.6. Thiazolidinediones藥物對於BeWo細胞之PPARα mRNA的影響 36 5.7. Thiazolidinediones藥物對於BeWo細胞之RXRα mRNA的影響 36 5.8. Pioglitazone對於細胞OCTN2、PPARα、RXRα蛋白質之影響 37 5.9. Troglitazone處理後MG132對於RXRα之影響 37 5.10. Thiazolidinediones藥物對於BeWo細胞之活性氧化物 (Reactive oxygen species,ROS)的影響 38 5.11. Troglitazone對於BeWo細胞之HIF-1α以及GLUT1 mRNA影響 38 5.12. Thiazolidinediones藥物對於HIF-1α 蛋白質表現之影響 38 5.13. Thiazolidinediones藥物對於BeWo細胞之MAPK表現之影響 39 5.14. Troglitazone對於HepG2細胞之影響 39 第6章 結果討論 55 第7章 結論 59 第8章 參考文獻 60 | |
dc.language.iso | zh-TW | |
dc.title | Thiazolidinediones影響胎盤第二新型有機陽離子轉運蛋白 (OCTN2)表現之機轉探討 | zh_TW |
dc.title | Mechanistic study of thiazolidinediones-mediated regulation of novel organic cation transporter type II (OCTN2) in BeWo cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 99-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 顧記華,陳美如,楊家榮 | |
dc.subject.keyword | 胎盤,第二新型有機陽離子轉運蛋白,thiazolidinediones,MAPK, | zh_TW |
dc.subject.keyword | placenta,OCTN2,thiazolidinediones,MAPK, | en |
dc.relation.page | 68 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2011-06-25 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 藥學研究所 | zh_TW |
顯示於系所單位: | 藥學系 |
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