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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 孔繁璐(Fan-Lu Kung) | |
| dc.contributor.author | Yi-Chi Tseng | en |
| dc.contributor.author | 曾奕淇 | zh_TW |
| dc.date.accessioned | 2021-06-13T03:48:15Z | - |
| dc.date.available | 2011-08-03 | |
| dc.date.copyright | 2006-08-03 | |
| dc.date.issued | 2006 | |
| dc.date.submitted | 2006-07-26 | |
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Characterization of organic cation/carnitine transporter family in human sperm. Biochem Biophys Res Commun. 306:121-8. | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/32419 | - |
| dc.description.abstract | 肉鹼 (L-carnitine) 在長鏈脂肪酸進入粒腺體以進行貝他氧化 (beta-oxidation) 反應及維持細胞內輔酵素A (CoA) 的恆定性上扮演了重要的角色。由於胎兒生合成肉鹼的能力尚未成熟因此需要主動獲取肉鹼以維持正常的功能。已知肉鹼主要是經由第二新型有機陽離子轉運蛋白 (organic cation transporter novel type II) 進入胎兒內。這種轉運蛋白對肉鹼有高親合力且其對肉鹼之攝取現象是依鈉性的。為了研究胎盤融合化的過程對第二新型有機陽離子轉運蛋白的表現以及功能之影響，實驗中使用源於人類絨毛膜癌細胞的BeWo細胞，BeWo細胞可藉由forskolin的刺激而產生融合化的現象，因此可作為一個胎盤的in vitro模式。
在現階段的實驗結果中發現當BeWo細胞加入100 uM的forskolin產生融合化的現象時，利用反轉錄－聚合酶連鎖反應進行分析，可以觀察到第二新型有機陽離子轉運蛋白的轉錄量有降低之情形。在以氚標幟之肉鹼觀察BeWo細胞對肉鹼的攝取量時，可以得知BeWo細胞攝取肉鹼是依鈉性且會飽和的，其動力學參數為：Km = 27.1 ± 11.8 uM，Vmax = 702 ± 132 pmol/30 min/mg protein，並帶有一個不飽和常數k，其值為6.02 ± 1.21 uL/30 min/mg protein，而在加入forskolin後這些動力學參數的值均有改變 (Km = 72.6 ± 5.4 uM，Vmax = 1805 ± 420 pmol/30 min/mg protein，k = 2.25 ± 0.59 uL/30 min/mg protein)，因此可以推斷在加入forskolin後所引起的融合化會使第二新型有機陽離子轉運蛋白的表現以及作用有所改變。另外我們亦探討一些藥物在forskolin所引起的BeWo細胞融合化中對肉鹼的半數抑制濃度 (IC50) 是否會改變以了解藥物對第二新型有機陽離子轉運蛋白之作用是否會受細胞融合化所影響。 | zh_TW |
| dc.description.abstract | L-carnitine is important for beta-oxidation of fatty acids and intracellular coenzyme A homeostasis. Given that the carnitine biosynthesis is immature in fetal organisms, it is pivotal for fetal organism to acquire carnitine to maintain normal functions. It is known that carnitine is mainly transported across placenta via organic cation transporter novel type II (OCTN2), a high affinity and sodium dependent carnitine transporter. BeWo cell is derived from human choriocarcinoma and can be induced to differentiate into syncytiotrophoblast by forskolin and has therefore been used as an in vitro placenta model. In the current study, BeWo treated with forskolin was used as a model for syncytiotrophoblast. Results from RT-PCR analysis showed that the expression level of OCTN2 was downregulated after 100 uM forskolin treatment. Uptake of 3H-labeled L-carnitine was sodium dependent and saturable (Km = 27.1 ± 11.8 uM, Vmax = 702 ± 132 pmol/30 min/mg protein) with a non-saturable constant k equals to 6.02 ± 1.21 (uL/30 min/mg protein). The values of kinetic parameters changed after forskolin treatment (Km = 72.6 ± 5.4 uM, Vmax = 1805 ± 420 pmol/30 min/mg protein, k = 2.25 ± 0.59 uL/30 min/mg protein). These results suggest that the expression level and function of OCTN2 are both changed during syncytialisation. IC50 for several drugs of carnitine uptake were also determinated to understand the effect on drug-OCTN2 interaction during syncytialisation induced by forskolin. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-13T03:48:15Z (GMT). No. of bitstreams: 1 ntu-95-R93423001-1.pdf: 1242969 bytes, checksum: 4f24e59369275ec0d604f184545a121a (MD5) Previous issue date: 2006 | en |
| dc.description.tableofcontents | 第一章 緒論 1
一、肉鹼 (carnitine) 1 二、與肉鹼相關的轉運蛋白 1 三、有機陽離子轉運蛋白 (organic cation transporter) 2 四、第二新型有機陽離子轉運蛋白 3 五、胎盤與胎盤屏障 4 六、胎盤細胞融合化 (syncytialisation) 5 七、第二新型有機陽離子轉運蛋白與藥物之關係 6 第二章 實驗目的 12 第三章 實驗材料 13 一、BeWo細胞培養 13 二、絨毛性腺激素 (β-hCG) 濃度測定 14 三、蛋白質濃度測定 (Bio-Rad DC protein assay) 14 四、肉鹼在BeWo細胞的攝取量研究 (uptake syudy) 、Transepithelial electrical resistance (TEER) 及BeWo細胞對肉鹼的穿透性 (transport study) 14 五、反轉錄及聚合酶鏈鎖反應分析 (reverse transcription polymerase chain reaction analysis，RT-PCR analysis) 15 六、藥物在BeWo細胞中對肉鹼的半數抑制濃度 (50% inhibitory concentration) 研究 18 七、其 他 18 第四章 實驗方法 20 一、BeWo細胞培養 20 二、絨毛性腺激素 (β-hCG) 濃度測定 20 三、蛋白質濃度測定 (Bio-Rad DC protein assay) 21 四、肉鹼在BeWo細胞的攝取量研究 22 五、Transepithelial electrical resistance (TEER) 23 六、BeWo細胞對肉鹼的穿透性試驗 (transport study) 23 七、反轉錄及聚合酶鏈鎖反應分析 (reverse transcription polymerase chain reaction analysis，RT-PCR analysis) 24 八、藥物在BeWo細胞中對肉鹼的半數抑制濃度 (50% inhibitory concentration) 研究 26 九、數據分析 27 第五章 實驗結果 29 第六章 結果討論 33 第七章 結 論 38 參考文獻 原始數據 圖目錄 圖一 肉鹼在脂肪酸氧化作用中的循環 8 圖二 人類SLC22A轉運蛋白家族之種系發生樹 (phylogenetic tree) 8 圖三 人類第二新型有機陽離子轉運蛋白 (hOCTN2) 之結構預測示意圖 9 圖四 第二新型有機陽離子轉運蛋白之突變位置與結構相對關係示意圖 9 圖五 BeWo細胞培養在細胞培養盤內槽 (cell culture insert) 中的TEER值 39 圖六 在BeWo細胞進行肉鹼穿透性試驗所得的肉鹼之穿透量對時間的關係圖 40 圖七 在BeWo細胞進行肉鹼穿透性試驗所得之肉鹼permeability對時間的關係 41 圖八 BeWo細胞在100μM forskolin以及溶媒(DMSO)處理後的β-hCG分泌量與時間關係圖 42 圖九 藉反轉錄－聚合酶連鎖反應放大GAPDH、syncytin和SLC22A5的mRNA與PCR循環數之關係 43 圖十 BeWo細胞在經由forskolin處理後利用反轉錄－聚合酶連鎖反應來觀察GAPDH的變化量之洋菜膠電泳及分析圖 44 圖十一 BeWo細胞在經由forskolin處理後利用反轉錄－聚合酶連鎖反應來觀察syncytin的變化量之洋菜膠電泳及分析圖 45 圖十二 時間和肉鹼攝取量在BeWo細胞中的關係圖 46 圖十三 BeWo細胞在經由forskolin處理後利用反轉錄－聚合酶連鎖反應來觀察SLC22A5的變化量之洋菜膠電泳及分析圖 47 圖十四 肉鹼濃度和肉鹼攝取量在BeWo細胞中的關係圖 48 圖十五 肉鹼濃度和肉鹼攝取量在經過forskolin處理過的BeWo細胞中之關係圖 49 圖十六 肉鹼濃度和肉鹼攝取量在forskolin處理的BeWo細胞中與對照之比較圖 50 圖十七 BeWo細胞在有無forskolin處理之肉鹼攝取在不同抑制劑存在下的百分比研究 51 表目錄 表一 SLC22A轉運蛋白家族之組織分布及功能特性表 10 表二 會造成原發性肉鹼缺乏症的第二新型有機陽離子轉運蛋白之突變位置表 11 表三 BeWo細胞在有無forskolin處理時的動力學參數值對照表 52 表四 各抑制劑對有無forskolin處理之BeWo細胞的肉鹼半數抑制濃度 53 | |
| dc.language.iso | zh-TW | |
| dc.title | 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 | zh_TW |
| dc.title | EFFECT OF FORSKOLIN ON THE EXPRESSION AND
FUNCTION OF ORGANIC CATION TRANSPORTER NOVEL TYPE II (OCTN2) IN HUMAN CHORIOCARCINOMA BeWo CELLS | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 94-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 林君榮(Chun-Jung Lin) | |
| dc.contributor.oralexamcommittee | 李財坤(Tsai-Kun Li),黃麗華(Lih-Hwa Hwang) | |
| dc.subject.keyword | 胎盤,第二新型有機陽離子轉運蛋白,細胞融合化, | zh_TW |
| dc.subject.keyword | OCTN2,Placenta,BeWo,syncytialisation,forskolin, | en |
| dc.relation.page | 67 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2006-07-26 | |
| 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 | |
文件中的檔案：
| 檔案 | 大小 | 格式 | |
|---|---|---|---|
| ntu-95-1.pdf 目前未授權公開取用 | 1.21 MB | Adobe PDF |
系統中的文件，除了特別指名其著作權條款之外，均受到著作權保護，並且保留所有的權利。