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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 | Ting-Ting Chang | en |
dc.contributor.author | 張婷婷 | zh_TW |
dc.date.accessioned | 2021-06-15T01:33:50Z | - |
dc.date.available | 2012-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-20 | |
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A. 92, 7921-7925. 黃富德 人類絨毛膜癌細胞 (BeWo) syncytialization中第二新型有機陽離子轉運蛋白 (OCTN2) 表現與功能機制之探討 (2008) 國立台灣大學醫學院藥學研究所碩士論文 簡郡緯 缺氧調控CD151表現對癌細胞附著能力的影響之探討 (2007)國立成功大學生理學研究所碩士論文 蘇怡寧 胎盤生長因數之功能分析及臨床應用 (2003) 國立台灣大學醫學院臨床醫學研究所博士論文 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/43035 | - |
dc.description.abstract | 人類胎盤中融合滋養層細胞 (syncytiotrophoblast) 是母親與胎兒間物質交換的介面。根據許多研究顯示,缺氧情況下融合滋養層細胞融合化 (syncytialization)的過程會受到抑制並且與懷孕時期所產生的嚴重併發症子癲前症 (pre-eclampsia)有關。肉鹼 (carnitine) 在脂肪酸代謝中能夠將長鏈脂肪酸運送進入到粒線體中進行貝他氧化作用 (β-oxidation) 來產生細胞所需之能量,然而在胎兒發育過程中因無法自行合成肉鹼,必須透過胎盤運輸由母親提供給胎兒利用。已知胎盤上第二新型有機陽離子轉運蛋白 (OCTN2) 對於運送肉鹼具有很高的親和力,有研究顯示患有子癲前症的婦女,血漿中肉鹼的含量高於正常懷孕婦女將近50 %,因此本實驗的目的在於探討低氧對於人類胎盤絨毛膜癌細胞 (BeWo) OCTN2表現的影響與其調控機轉之研究。
本實驗中將細胞培養在0.5 % O2來模擬低氧的環境,透過西方墨點法來觀察融合化前後BeWo細胞膜上Syncytin、OCTN2與PDZK1蛋白與在低氧環境中變化的情形。除此之外,也藉由觀察細胞核蛋白HIF-1α、HIF-2α、PPARα與RXRα的改變情形來推測低氧環境下對於融合前BeWo細胞膜上OCTN2調控的機制。結果顯示在forskolin誘導下syncytin與OCTN2的表現皆增加,然而在低氧環境下則是下降的,PDZK1的表現在誘導融合化後顯著的下降,低氧下卻有上升的趨勢。HIF-1α和HIF-2α蛋白在4小時被誘導增加,但24小時後隨即下降。PPARα與RXRα兩者蛋白的表現在24小時顯著下降。另一方面,在給予PPARα專一的agonist WY14643處理後OCTN2的表現是上升的,並且實驗中發現在低氧環境下也可誘導OCTN2的表現增加。接著進一步利用氚標定之肉鹼,來探討低氧與WY14643處理後OCTN2轉運肉鹼之情形,結果中發現給予WY14643處理後能夠提升OCTN2轉運肉鹼的能力,然而於低氧環境下則是下降的。最後利用免疫組織化學染色的發法觀察人類胎盤組織的結果,發現在患有子癲前症婦女的胎盤上PPARα和OCTN2蛋白的表現皆是下降的。 總結上面的結果,在低氧環境下BeWo細胞融合化的過程和OCTN2的表現以及肉鹼運轉的能力是受到抑制的,根據人類胎盤組織染色的結果我們推論OCTN2的表現下降可能為造成子癲前症婦女體內肉鹼累積的原因,此外本實驗也提供證據顯示PPARα為影響OCTN2的表現的重要因子,然而是否有其他調控因子參與其中仍需要做更進一步的探討。 | zh_TW |
dc.description.abstract | The syncytiotrophoblast of human placenta is an important interface mediating substance transfer between the mother and the fetus. Preeclampsia, a serious complication during pregnancy, is associated with impaired syncytialization. Carnitine is responsible for the transport of long-chain fatty acids into mitochondria, which is then undergoing β-oxidation for cellular energy production. However, 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 human placenta. It was reported that plasma carnitine concentrations in pregnant women with preeclampsia increased about 50 % compared with healthy pregnant women. Hence, the aim of this study was to investigate the effects of hypoxic condition on the regulation of OCTN2 in human choriocarcinoma BeWo cells.
BeWo cells were cultured under 0.5 % O2 for mimicking hypoxic condition. The protein expression of syncytin, OCTN2, and PDZK1 in crude membrane of BeWo cells was analyzed by Western blot analysis, and the nuclear expression of HIF-1α, HIF-2α, PPARα and RXRα was also measured to explore possible mechanisms in regulating OCTN2 under hypoxic condition. Under forskolin-induced syncytialization, syncytin and OCTN2 were increased, whereas both of them were decreased in hypoxic condition. PDZK1 was significantly downregulated after syncytialization, whereas it was slightly increased under hypoxia. HIF-1α and HIF-2α were upregulated at 4 hours and then decreased at 24 hours after hypoxia treatment. Both of PPARα and RXRα were significantly downregulated at 24 hours. However, OCTN2 expression was increased upon treatment PPARα agonist, WY14643. The activation of OCTN2 expression was also upregulated by WY14643 under hypoxic condition. Afterward, cellular uptake of 3H-labeled L-carnitine was measured after hypoxia and WY14643 treatment. The results showed that the values of carnitine uptake was increased upon WY14643 treatment, however it was decreased under hypoxia condition. We also observed human normal and preeclamptic placental tissue by immunohistochemistry. Both of OCTN2 and PPARα protein expression were decreased in human preeclamptic placentas. In conclusion, the process of syncytialization, OCTN2 protein expression and carnitine uptake was inhibited under hypoxic condition. According to the results of immunohistochemistry, we speculate the plasma carnitine concentrations accumulation in preeclamptic pregnant women can be due to the downregulated OCTN2 expression. Furthermore, we also provide evidences that PPARα is one of the important factors that regulate the expression of OCTN2. If there are any other factors involved in regulating OCTN2 should be verified. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:33:50Z (GMT). No. of bitstreams: 1 ntu-98-R96423012-1.pdf: 2678410 bytes, checksum: ddab08f5f3d9e1da1d41de299a014b39 (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 中文摘要……………………………………………………………………i
英文摘要…………………………………………………………………..iii 第一章 序論……………………………………………………………...1 1.1胎盤與血胎屏障……………………………………………………..1 1.2胎盤細胞的融合……………………………………………………..2 1.3胎盤發育時期氧氣含量的變化與子癲前症....………………………….3 1.4缺氧誘導因數 (HIF) …………………………………………………4 1.5肉鹼 (L-carnitine)與第二新型有機陽離子轉運蛋白 (OCTN2) ……….…5 1.6過氧化體增殖活化受體 (PPARs)..………..………….………..………6 1.6.1過氧化體增殖活化受體 (PPARα)...........................7 1.7 PDZK1 .........................................................8 第二章 實驗目的 .............................................16 第三章 實驗材料 ...............................................17 3.1 BeWo細胞培養............................................17 3.1.1試劑....................................................................................17 3.1.2材料與設備..........................................................................17 3.2 BeWo細胞低氧培養以及藥物處理 ..................................................18 3.2.1試劑....................................................................................18 3.2.2設備....................................................................................18 3.3臨床人類胎盤組織收集 ......................................18 3.4蛋白質的測定 (Bio-Rad DC protein assay) .......................................18 3.4.1材料與設備..........................................................................18 3.5細胞膜的萃取 ................................................................................18 3.5.1試劑....................................................................................18 3.5.2材料與設備 .........................................................................19 3.6核蛋白的萃取 ................................................................................19 3.6.1試劑....................................................................................19 3.6.2材料與設備....................................................................20 3.7西方墨點法 ...................................................................................20 3.7.1試劑....................................................................................20 3.7.2一級抗體 ..............................................................................21 3.7.3二級抗體 ............................................................................21 3.7.4材料與設備........................................................................22 3.8肉鹼在BeWo細胞的攝取量研究 (uptake study) ................................. 22 3.8.1試劑....................................................................................22 3.8.2材料與設備..........................................................................23 3.9免疫組織化學染色 .............................................................................23 3.9.1試劑....................................................................................23 3.9.2一級抗體 ..............................................................................23 3.9.3二級抗體 ..............................................................................23 3.10其他 ...........................................................................................23 3.10.1其他儀器.......................................................................... 23 第四章 實驗方法 ..............................................24 4.1 BeWo細胞株培養..........................................24 4.2 藥物與低氧處理BeWo細胞株................................24 4.3 萃取細胞蛋白質 .............................................25 4.3.1膜蛋白的萃取 ........................................25 4.3.2核蛋白的萃取 .......................................25 4.4 蛋白質濃度測定 ............................................26 4.5西方墨點法 .................................................26 4.6肉鹼在BeWo細胞的攝取量研究................................27 4.6.1單點肉鹼攝取量研究..................................27 4.7免疫組織化學染色............................................28 4.8數據分析.................................................29 第五章 實驗結果 ..............................................30 5.1正常與低氧環境下Forskolin誘導BeWo融合化之研究 ............30 5.2 BeWo誘導融合化後OCTN2蛋白於正常與低氧環境下細胞膜上變化之研究 ....................................................30 5.3 BeWo誘導融合化後PDZK1蛋白於正常與低氧環境下細胞膜上變化之研究 ........................................................30 5.4 低氧環境下BeWo細胞核內HIF-1α、HIF-2α、PPARα和RXRα蛋白變化之研究 .............................................................................31 5.5 BeWo細胞處理PPARα agonist WY14643前後OCTN2蛋白於細胞膜上變化之研究 ....................................................31 5.6 低氧環境下BeWo細胞處理PPARα agonist WY14643前後OCTN2蛋白於細胞膜上變化之研究 ..............................................32 5.7在BeWo細胞中肉鹼攝取量之研究..............................32 5.8低氧環境下人類胎盤組織PPARα和OCTN2蛋白變化之研究..................................................................................................32 第六章 結果討論 ................................................41 第七章 結論.........................................................46 第八章 參考文獻...............................................47 附錄.........................................................57 原始數據...............................................................................59 | |
dc.language.iso | zh-TW | |
dc.title | 低氧環境對人類胎盤絨毛膜癌BeWo細胞上第二新型有機陽離子轉運蛋白(OCTN2)表現影響之研究 | zh_TW |
dc.title | Effect of hypoxia on the expression of organic cation transporter novel type II (OCTN2) in human choriocarcinoma BeWo cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃敏銓,徐明洸,符文美 | |
dc.subject.keyword | 第二新型有機陽離子轉運蛋白,人類胎盤絨毛膜癌BeWo細胞,低氧,細胞融合化, | zh_TW |
dc.subject.keyword | OCTN2,BeWo cells,hypoxia,syncytialization, | en |
dc.relation.page | 62 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-20 | |
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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