請用此 Handle URI 來引用此文件：
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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 王淑美 | |
| dc.contributor.author | Yen-Tung Lin | en |
| dc.contributor.author | 林姸彤 | zh_TW |
| dc.date.accessioned | 2021-06-16T13:02:46Z | - |
| dc.date.available | 2023-08-05 | |
| dc.date.copyright | 2013-09-24 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-08-06 | |
| dc.identifier.citation | 參考文獻
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61425 | - |
| dc.description.abstract | 細胞外酸性pH (pHe) 為腫瘤細胞生長環境的特徵之一，並且酸性pHe會影響腫瘤細胞移行及侵犯能力。我們研究室先前已證實肝癌細胞(HepG2)經酸化處理後，藉由Src活化並破壞adherens junction(AJ)，而使細胞分離，促進細胞移行能力。本實驗更進一步探討酸性pHe是否經由ROS，而增加癌細胞移行能力。實驗結果顯示，對照組pH 7.4中細胞呈緊密接合的群聚狀態，pH 6.6處理後細胞變成分散且扁平，伴隨細胞移行能力增加。利用dichlorofluorescein diacetate(DCFDA)及dihydroethidium(DHE)兩種指示劑以流式細胞儀檢查，發現pH 6.6處理後細胞內ROS量明顯上升，進而使用專一偵測粒線體內ROS螢光探針MitosoxTM，也可測得ROS量增加，證明ROS可能來自於粒線體。而這些ROS可以刺激細胞移行，因為抗氧化劑dithiothreitol(DTT)能阻止因pH 6.6處理引起的細胞移行。同時，pH 6.6處理30分鐘後ERK的磷酸化增加，持續至90分鐘為止。其次，利用抑制劑U0126抑制MEK及siRNA技術降低ERK的蛋白質表現量，皆可阻止因pH 6.6處理引起的細胞移行。此外DTT能有效抑制ERK磷酸化，說明ROS為ERK的上游分子。以pH 6.6處理後，觀察到黏附斑聚集在細胞膜邊緣且原先隨意分佈在細胞質中的微絲重新排列成平行方向性的張力絲。利用西方墨點法證明pH 6.6處理促使FAK磷酸化。此外，FAK抑制劑PF573228與siRNA技術減少FAK蛋白質表現量，皆能有效地減緩細胞移行，表示FAK參與調控酸性環境中細胞移行的機制。而利用DTT預處理可以減少因酸性刺激而產生的ERK和FAK磷酸化，說明ROS為這兩者的上游分子。進一步利用U0126及siERK預處理，顯示兩者皆能有效抑制FAK磷酸化且阻止細胞移行，表示存在ERK-FAK這條訊息傳遞路徑。另外，pH 6.6處理會促使Src與p130CAS活化，且Src抑制劑PP2能干擾酸性刺激而誘導的張力絲生成，意味著Src-p130CAS訊息傳遞路徑有參與調控張力絲形成。總結而言，本研究提供另一個不同的訊息傳遞路徑，說明肝癌細胞在酸性環境的刺激後，是藉由ROS/ERK/FAK訊息傳遞達到促進細胞移行。 | zh_TW |
| dc.description.abstract | An acidic extracellular pH (pHe) is a feature of the solid tumor microenvironment, which contributes to cell migration and invasion of tumor cells. Our previous study has demonstrated that acidic pHe disrupts the integrity of adherens junction of hepatocellular carcinoma (HepG2) by activation of Src, and promotes migration by cell dispersion. In this study, we investigated whether reactive oxygen species (ROS), a signaling messenger, initiated the signaling for acidic pHe-induced cell migration. Our results showed that incubation of HepG2 cells in pH 6.6 culture medium induced HepG2 cells from tight cluster to a dispersed and flattened cell profile, and this change was accompanied by an increase in cell migration ability. By flow cytometry, pH 6.6 treatment induced higher ROS levels compared to those at pH 7.4. Furthermore, ROS production mainly came from mitochondria by flow cytometry with MitosoxTM , mitochondria superoxide indicator. pH 6.6- induced-ROS increased cell migration, since an antioxidant dithiothreitol (DTT) prevented this effect. Extracellulr signal-regulated kinase (ERK) phosphorylation increased at 30 minutes after pH 6.6 stimulation, and sustained up to 90 minutes. Inhibition of ERK by a mitogen-activated protein kinase (MEK) inhibitor, U0126, or by siERK abrogated pH 6.6- induced cell migration of HepG2 cells. In addition, DTT blocked pH 6.6-induced phosophorylation of ERK. This suggests that ROS is the upstream event of ERK. In response to pH 6.6 treatment, focal adhesions clustered at peripheral cell membrane and randomly-distributed microfilaments were rearranged into parallel – oriented stress fibers. Western blotting analyses demonstrated that FAK was phosphorylated by pH 6.6 treatment. Furthermore, FAK played a key role in cell migration, since inhibition of FAK by a FAK inhibitor , PF573228, or by siFAK effectively prevented pH 6.6-induced cell migration. DTT pretreatment abrogated pH 6.6-induced phosophorylation of ERK and FAK. It implicates that ROS is the upstream event of both ERK and FAK. Inhibition of ERK by U0126 and siERK blocked pH 6.6- induced phosphorylation of FAK and retarded cell migration, indicating the presence of the ERK-FAK pathway. Besides, Src and p130CAS were activated by pH 6.6 treatment, and PP2 pretreatment disturbed pH 6.6-induced stress fiber formation, suggesting the involvement of the Src-p130CAS pathway in this event. In conclusion, this study characterize a novel signaling way responsible for enhancing the migratory behavior of HepG2 cells via the ROS- ERK- FAK signaling in an acidic microenvironment. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T13:02:46Z (GMT). No. of bitstreams: 1 ntu-102-R00446005-1.pdf: 4099183 bytes, checksum: 1bf7f7037b33808a69e3c28943605987 (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 誌謝 ………………………………………… P. i
中文摘要 ……………………………………… P. ii∼P. iii 英文摘要 ……………………………………… P. iv∼P. v 前言 …………………………………………… P.2∼P.9 材料與方法 …………………………………… P.10∼P.17 結果 …………………………………………… P.18∼P.27 討論 …………………………………………… P.28∼P.36 參考文獻 ……………………………………… P.37∼P.43 圖片與圖片說明 ……………………………… P.44∼P.65 附錄、模式圖 ………………………………….. P.66 | |
| dc.language.iso | zh-TW | |
| dc.subject | 肝癌細胞 | zh_TW |
| dc.subject | 酸性酸鹼值 | zh_TW |
| dc.subject | 細胞移行 | zh_TW |
| dc.subject | 活性氧 | zh_TW |
| dc.subject | 黏附斑 | zh_TW |
| dc.subject | 張力絲 | zh_TW |
| dc.subject | focal adhesion | en |
| dc.subject | HepG2 | en |
| dc.subject | acidic pH | en |
| dc.subject | cell migration | en |
| dc.subject | reactive oxygen species | en |
| dc.subject | stress fiber | en |
| dc.title | 細胞外酸性酸鹼值經ROS-ERK-FAK 訊息傳遞路徑促進HepG2細胞移行 | zh_TW |
| dc.title | An Acidic Extracellular pH Promotes Cell Migration of HepG2 via the ROS-ERK-FAK Signal Pathway | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 吳建春,陳玉怜,鄭瓊娟,龔秀妮 | |
| dc.subject.keyword | 肝癌細胞,酸性酸鹼值,細胞移行,活性氧,黏附斑,張力絲, | zh_TW |
| dc.subject.keyword | HepG2,acidic pH,cell migration,reactive oxygen species,focal adhesion,stress fiber, | en |
| dc.relation.page | 66 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2013-08-06 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 解剖學暨細胞生物學研究所 | zh_TW |
| 顯示於系所單位： | 解剖學暨細胞生物學科所 | |
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