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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 | Fu-De Huang | en |
dc.contributor.author | 黃富德 | zh_TW |
dc.date.accessioned | 2021-06-08T07:14:18Z | - |
dc.date.copyright | 2008-08-08 | |
dc.date.issued | 2008 | |
dc.date.submitted | 2008-07-29 | |
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(1999) cAMP-induced phosphorylation and inhibition of Na+/H+ exchanger 3 (NHE3) are dependent on the presence but not the phosphorylation of NHE regulatory factor. The Journal of Biological Chemistry 274, 24753-24758. 曾奕淇 探討Forskolin在人類絨毛膜癌細胞 (BeWo) 中對第二新型有機陽離子轉運蛋白之表現及作用的影響 (2006) 國立台灣大學藥學研究所碩士論文 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/26539 | - |
dc.description.abstract | 人類胎盤中融合滋胚層細胞為母親與胎兒間交換藥物及養分的界面,而胎盤細胞融合化對於胎盤正常功能發育中為一重要過程。已知肉鹼為負責將體內長鍊脂肪酸運送至粒線體中以進行貝他氧化作用 (β-oxidation) 以產生細胞所需之能量。然而在胎兒發育的過程中,胎兒體內自行合成肉鹼的能力尚未成熟,因此須仰賴母親血液中之肉鹼穿過胎盤以提供胎兒所需。由於肉鹼對於胎兒在發育的過程中的不可或缺,因此本實驗之目的在於探討在懷孕初期胎盤細胞融合化過程中肉鹼轉運蛋白又名為第二新型有陽離子轉運蛋白 (OCTN2) 之表現與功能改變原因之探討。forskolin已經被報導可以順利誘導BeWo細胞產生融合化現象,因此在此實驗中我們使用其作為模擬胎盤細胞融合化過程之體外模式。
實驗過程中我們利用反轉錄聚合脢鏈鎖分析及萃取細胞膜及刷狀外緣絨毛膜之部分並觀察syncytin之mRNA與蛋白質表現來作為BeWo細胞融合化之確認,並且觀察OCTN2、PDZK1、PDZK2、NHERF1與NHERF2蛋白在融合過程中之改變情形,接著進一步利用氚標定之肉鹼,來探討融合過程中OCTN2轉運肉鹼之動力學改變情形。結果顯示forskolin可以成功誘導細胞之syncytin的mRNA與蛋白表現量增加,表示細胞順利誘導進行融合,並且在長時間forskolin作用下,發現OCTN2於細胞膜與刷狀絨毛膜上表現量皆顯著增加,然而其對於肉鹼之親和力顯著下降 (Km增加),但是肉鹼轉運能力 (Vmax) 卻沒有改變,而PDZK1與NHERF1於細胞膜與刷狀絨毛膜上表現量則在細胞誘導分化後顯著下降。 總結以上之實驗結果,我們發現在forskolin的誘導細胞融合過程中,OCTN2的蛋白表現與功能確實會受到影響,OCTN2之功能調控因子PDZK1表現量下降,導致OCTN2對於肉鹼之轉運能力下降,但是由於在細胞膜與刷狀絨毛膜上蛋白表現量增加,導致在細胞融合過程中其肉鹼攝取能力產生彌補,以致肉鹼攝取維持一定,目前已知PDZK1為許多轉運蛋白功能表現之調控因子,然而對於PDZK1在細胞融合過程中造成其他轉運蛋白功能之影響尚須做更進一步的探討。 | zh_TW |
dc.description.abstract | The syncytiotrophoblast of human placenta provides an essential surface in mediating the transfer of drugs and nutrients between the mother and the fetus. Syncytialization is an important process for the development of functional placenta. Carnitine is important for the transport of long-chain fatty acid to mitochondria, which is then subjected to β-oxidation. However, the carnitine biosynthesis in fetal organisms is immature and placental transfer of carnitine from maternal blood is required. Given the essential role of carnitine for fetal development, the purpose of the current study was to investigate the effects of syncytialization on the expression and function of OCTN2, a high-affinity carnitine transporter. The forskolin induced syncytialization of BeWo cells was used as an in vitro model for placental trophoblast in this study.
Crude membrane fraction and brush border membrane were isolated. The mRNA and protein expression of syncytin were analyzed to test whether forskolin treatment can induce syncytialization in BeWo cells. The change in the expression of OCTN2 and adapter proteins, PDZK1, PDZK2, NHERF1 and NHERF2, under forskolin treatment was analyzed by Western blot. Afterward, cellular uptake of 3H-labeled L-carnitine was measured and kinetic analysis was performed. The results showed that forskolin can increase the mRNA and protein expression of syncytin. Protein expression of OCTN2 is increased under long-term forskolin treatment. However, the kinetic study showed that the Vm values of carnitine uptake remained unchanged and the Km values increased. Both PDZK1 and NHERF1 protein expression were decreased after forskolin treatment. In conclusion, protein and function of OCTN2 can be regulated during syncytialization. OCTN2 protein is up-regulated, whereas PDZK1, the functional regulator of OCTN2, is decreased and result in the unchanged Vmax. The regulation of PDZK1 on other transporters during syncytialization should be further varified. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T07:14:18Z (GMT). No. of bitstreams: 1 ntu-97-R95423018-1.pdf: 1312893 bytes, checksum: 9c3901d923905f8de763815021631e6f (MD5) Previous issue date: 2008 | en |
dc.description.tableofcontents | 目錄 i
圖目錄 v 表目錄 vii 表目錄 vii Abstract viii 中文摘要 ix 第1章 緒論 1 1.1. 胎盤與胎盤屏障 1 1.2. 胎盤細胞融合 2 1.3. 肉鹼 (L-Carnitine) 3 1.4. 有機陽離子轉運蛋白 4 1.4.1. 第一新型有機陽離子轉運蛋白 (OCTN1) 5 1.4.2. 第二新型有機陽離子轉運蛋白 (OCTN2) 5 1.5. PDZ蛋白 6 1.5.1. PDZ adaptor protein 6 1.6. Caveolae 8 第2章 實驗目的 9 第3章 實驗材料 10 3.1. BeWo細胞培養 10 3.1.1. 試劑 10 3.1.2. 材料與設備 10 3.2. 蛋白質濃度測定 (Bio-Rad DC protein assay) 10 3.2.1. 材料與設備 10 3.3. 肉鹼在BeWo細胞的攝取量研究 (uptake study) 11 3.3.1. 試劑 11 3.3.2. 材料與設備 11 3.4. 反轉錄聚合酶鏈鎖反應分析 (reverse transcription polymerase chain reaction analysis,RT-PCR) 11 3.4.1. 試劑 11 3.4.2. 材料與設備 13 3.5. 細胞膜與刷狀外緣絨毛膜萃取 13 3.5.1. 試劑 13 3.5.2. 緩衝液配方 14 3.5.3. 材料與設備 14 3.6. 蔗糖梯度離心 14 3.6.1. 試劑 14 3.6.2. 材料與設備 15 3.7. 西方墨點法 15 3.7.1. 試劑 15 3.7.2. 緩衝液配方 16 3.7.3. 一級抗體 17 3.7.4. 二級抗體 17 3.7.5. 材料與設備 17 3.8. 免疫螢光染色 18 3.8.1. 試劑 18 3.8.2. 一級抗體 18 3.8.3. 二級抗體 18 3.8.4. 設備 18 3.9. 其他 18 3.9.1. 緩衝液配方 18 3.9.2. 其他儀器 19 第4章 實驗方法 20 4.1. BeWo細胞株培養 20 4.2. 藥物處理BeWo細胞株 20 4.3. 膜蛋白萃取 20 4.3.1. Crude membrane extraction 21 4.3.2. Brush border membrane extraction 21 4.3.3. Lipid raft extraction 21 4.4. 鹼性磷酸酶測定 22 4.5. 蛋白質濃度測定 23 4.5.1. 藥物攝取實驗之蛋白濃度測定 23 4.5.2. 膜蛋白之蛋白質濃度測定 23 4.6. 反轉錄及聚合酶鏈鎖反應分析 24 4.6.1. RNA萃取 24 4.6.2. 反轉錄反應 24 4.6.3. DNA聚合酶鏈鎖反應 25 4.6.4. 洋菜膠電泳分析 25 4.7. 西方墨點法 25 4.8. 免疫螢光染色 26 4.9. 肉鹼在BeWo細胞攝取量研究 27 4.9.1. 單點肉鹼攝取量之研究 27 4.9.2. 濃度相依性研究 27 4.10. 數據分析 28 第5章 實驗結果 29 5.1. Forskolin 誘導BeWo細胞分化之研究 29 5.2. 刷狀外緣絨毛膜 (BBM) 純化之結果 29 5.3. BeWo細胞誘導分化後OCTN2蛋白於細胞膜 (crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 29 5.4. BeWo細胞誘導分化後Caveolin-1蛋白於細胞膜上 (crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 30 5.5. BeWo細胞誘導分化後Caveolin-2蛋白於細胞膜 (crude membrane) 上變化之研究 30 5.6. BeWo細胞誘導分化後OCTN2與Caveolin-1關係之研究 31 5.7. OCTN2於BeWo細胞內分布情形之研究 31 5.8. 不同濃度及時間forskolin處理對於肉鹼攝取量之研究 31 5.9. BeWo細胞誘導分化後對於肉鹼攝取量之濃度相依性研究 32 5.10. BeWo細胞誘導分化後PDZK1蛋白於細胞膜 (crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 32 5.11. BeWo細胞誘導分化後PDZK2蛋白於細胞膜 (crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 33 5.12. BeWo細胞誘導分化後NHERF1蛋白於細胞膜(crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 33 5.13. BeWo細胞誘導分化後NHERF2蛋白於細胞膜 (crude membrane) 與刷狀外緣絨毛膜上 (BBM) 上變化之研究 34 第6章 結果討論 58 第7章 結論 66 參考文獻 67 原始數據 80 附錄 87 | |
dc.language.iso | zh-TW | |
dc.title | 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 | zh_TW |
dc.title | Investigation of mechanisms regulating protein expression and function of organic cation transporter novel type II (OCTN2) during syncytialization in human choriocarcinoma BeWo cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 96-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 孔繁璐,符文美,陳美如,徐明洸 | |
dc.subject.keyword | 人類絨毛膜癌細胞,第二新型有機陽離子轉運蛋白, | zh_TW |
dc.subject.keyword | BeWo,OCTN2, | en |
dc.relation.page | 91 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2008-07-30 | |
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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