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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 沈麗娟(Li-Jiuan Shen) | |
dc.contributor.author | Hui-An Sun | en |
dc.contributor.author | 孫慧恩 | zh_TW |
dc.date.accessioned | 2021-06-16T17:52:39Z | - |
dc.date.available | 2017-09-19 | |
dc.date.copyright | 2012-09-19 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-13 | |
dc.identifier.citation | 1. Lu, Y., J. Yang, and E. Sega, Issues related to targeted delivery of proteins and peptides. AAPS J, 2006. 8(3): p. E466-78.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64528 | - |
dc.description.abstract | 部分的蛋白質類藥物之作用目標位於細胞內,需要突破細胞膜的天然屏障,進到細胞內,才能發揮其效果。因此,開發合適的蛋白質胞內遞輸策略是一值得深入研究的課題。主動標靶遞送是一個新的藥物遞送策略,其概念在欲遞送的藥物或載體表面將某特定配體鍵結上,使整個遞送系統可被特定組織細胞表面的受器所辨認,並經由受體媒介的胞吞作用攝入細胞,增進欲遞送藥物或載體的臨床效果,且減少對其他組織所造成的副作用。肝臟所表現的 ASGPR (asialoglycoprotein receptor) 是一種可以專一性地辨識半乳糖基的膜蛋白,因此,我們嘗試在蛋白質上修飾半乳糖基,探討在這樣的修飾後,肝癌細胞對於蛋白質的攝取量是否能有所增加,並應用於蛋白質類藥物的胞內遞送。
我們初步利用細胞色素 C (cytC) 當作模式蛋白,利用還原胺化反應將 galactose-CHO 鍵結至 cytC 的氨基,得到 cytC-半乳糖接合體 (cytC-galactose conjugates; cytC-gal),並投予 HepG2 肝癌細胞株及 MCF7 乳腺癌細胞株,來評估 cytC-gal 之胞內遞送效果。我們將 cytC-gal 標記上 FTIC 螢光分子,以流式細胞儀來偵測細胞的攝取情形;並利用細胞存活率試驗 (MTT 試驗) 來了解鍵結不同半乳糖基數目的 cytC-gal 對細胞存活率之影響, 在 cytC-gal 的製備,隔夜反應 16 小時後即可鍵結 12 個半乳糖基,大幅地縮短了使用傳統糖基所需的反應時間。在細胞的攝取方面,將蛋白投予細胞一個小時後,在 HepG2 細胞株觀察到 cytC-FITC-gal8 的細胞攝取情形明顯高於 cytC-FITC (p < 0.001),但在 MCF7 細胞株則觀察不到這個現象。本實驗另外添加 ASGPR 的天然配體 (asialofetuin;ASF),藉由過量的 ASF 添加後對 cytC-FITC-gal8 攝取程度的抑制與否,推論 cytC-FITC-gal8 的攝取是否有經由 ASGPR。在 HepG2 細胞株,添加一倍莫耳濃度的 ASF,即可顯著地降低攝取 cytC-FITC-gal8 的細胞所占百分比,以及細胞平均螢光強度 (p < 0.001),證明 cytC-FITC-gal8 在 HepG2 細胞株大部分是藉由 ASGPR 而進入細胞。而在 MCF 7 細胞株,即使添加高達 30倍的 ASF,仍無法對 cytC-FITC-gal8 的攝取量有任何影響,證明 cytC-FITC-gal8 的細胞攝取與 ASGPR 可能無關。 在細胞存活率試驗中,我們藉由調整 galactose – CHO 與蛋白質的莫耳數比,分別鍵結了 5、8 以及 12 個半乳糖基。我們觀察到三種 cytC-gal 皆可降低 HepG2 及 MCF7 細胞株的存活率,所降低的程度與濃度呈正相關,間接表示半乳糖的修飾可將 cytC 遞送進細胞內引發細胞凋亡,並沒有呈現對細胞的選擇性。我們接下來投予相同酵素活性的 cytC-gal,希望藉此比較各種半乳糖鍵結量對胞內遞送的效果。然而,無論在 HepG2 或是 MCF7 細胞株,相較於接上 5 個半乳糖基而言,8 及 12 個半乳糖基的鍵結量並不會更進一步地使細胞存活率降低,間接表示未增加 cytC 的細胞攝取量。而為了要初步了解 cytC-gal 的細胞攝取機制,我們在細胞存活率試驗中投予 endosomal escape 試劑 chloroquine,希望能了解 cytC-gal 的細胞攝取是否有經過 endosome。結果顯示,在 HepG2 細胞株添加 chloroquine 會更進一步地降低細胞存活率,這或許代表 chloroquine 可以避免 endosome 內的 cytC-gal 被溶酶體分解,將有效的蛋白質釋出至細胞質,發揮藥效。而在另一個細胞株 MCF7,則觀察到 chloroquine 的併用並沒有對 cytC-gal 所造成的細胞生長抑制產生影響,間接代表 cytC-gal 在 MCF7 的細胞攝取可能沒有透過 endosome,而是走別的路徑。 基因重組精胺酸去亞胺酶 (recombinant arginine deiminase,rADI) 雖對大部分肝癌及黑色素瘤細胞具有抑制生長之作用,但仍有部分癌細胞對rADI具有抗性,如 HepG2 細胞株。根據本實驗室先前的經驗,曾利用細胞穿透胜肽攜帶 rADI 進入細胞,降低細胞內的精胺酸含量,改善對於 rADI 有抗性的問題。因此,我們最後將 galactose-CHO 鍵結至 rADI,希望改善 rADI 在 HepG2 細胞株的抗性。我們同樣利用細胞存活率試驗,結果顯示 rADI-gal 顯著地抑制了 HepG2 細胞的生長,間接代表將更多的 rADI 運送入 HepG2 細胞。 總結以上所有結果,利用 galactose-CHO 可將半乳糖基鍵結至 cytC 及 rADI 兩種蛋白質,並且在 HepG2 及 MCF7 細胞株均增進蛋白質藥物對細胞生長的抑制效果。在 HepG2 細胞株的細胞的攝取主要是藉由 ASGPR;而在 MCF7 細胞株的細胞攝取情形尚未被觀察到,因此也無法確定 cytC-gal 對 MCF7 細胞株生長抑制之原因。 | zh_TW |
dc.description.abstract | Many protein drugs need to overcome the cellular membrane barrier and be delivered intracellularly to exert their therapeutic action within the cell. Therefore, the development of a suitable strategy for intracellular protein delivery is a topic worth investigating. Active targeting delivery is one of the commonly used drug delivery strategy. Specific ligands are conjugated on the surface of the drug or carrier, and therefore enable the entire system to be recognized by a specific receptor expressed on the cellular surface of the target tissues and be taken into cells via receptor-mediated endocytosis. This strategy can further enhance the therapeutic efficacy of delivered drug or carrier, and at the same time reduce possible side effects on other tissues. Liver expresses the membrane protein ASGPR (asialoglycoprotein receptor) that can specifically recognize galactose residues. Therefore, we aim to modify protein with galactose ligand and then investigate the effect of galactose number on liver cellular uptake, and further apply this strategy to intracellular protein delivery.
First, we first used cytochrome C (cytC) as a model protein. Different numbers of galactose-CHO were conjugated to the amino groups on cytC via reductive amination to obtain cytC-galactose conjugates (cytC-gal). Hepatocellular carcinoma cell line, HepG2, and breast adenocarcinoma cell line, MCF7, were treated with cytC-gal conjugates to evaluate the intracellular delivery after galactose modification. CytC-gal were further labeled with FITC to detect cellular uptake via flow cytometry. We also performed MTT assay as the cell viability assay to investigate the effect of various cytC-gal conjugates on cell viability. Our results showed that it took 16 hours overnight to conjugate 12 galactose residues to cytC, which greatly reduced the reaction time compared with traditional method. In terms of cellular uptake, cellular uptake of cytC-FITC-gal8 in HepG2 was significantly higher than cytC-FITC (p < 0.001), but similar observation was not seen on MCF7. According to literature, liver tissues express a type of lectin named asialoglycoprotein receptor (ASGPR) that specifically recognizes glycoproteins containing terminal galactose or N-acetylgalactosamine residues. To understand if the uptake was ASGPR-mediated, we co-treated cells with excess asialofetuin (ASF), a native ligand for ASGPR. In HepG2 cells, co-incubation of cytC-FITC-gal8 with only 1 fold of ASF can significantly reduce both the percent of FITC-positive cells and fluorescence intensity (p <0.001), suggesting that ASGPR is a major pathway for cytC-FITC-gal8 cellular uptake in HepG2 cells. On the other hand, in MCF7, co-incubation of cytC-FITC-gal8 with 30 fold of ASF still had no effect on cellular uptake, implying that the cellular uptake of cytC-FITC-gal8 was not ASGPR-mediated. By adjusting the feed ratio of galactose-CHO to protein, we conjugated 5, 8 and 12 galactose residues. In cell viability experiments, we observed that a three cytC-gal conjugates can significantly reduce cell viability in both HepG2 and MCF7 cell lines in a dose-dependent manner. It indirectly indicated that galactose modification can enhance the intracellular delivery of cytC and induce cellular apoptosis. Cells were further treated with cytC-gal with identical enzyme activity to further investigate the effect of galactse residue numbers on intracellular delivery. However, we noticed that increasing galactose ligand from 5 to 8 and 12 did not further reduce cell viability in both cell lines, suggesting that the cellular uptake cannot be further enhanced either. We further treated cells with the endosomal escape reagent, chloroquine, to understand if the cellular uptake of cytC-gal was endosome-mediated. In HepG2 cells, it was found that chloroquine could further decrease the cell viability, suggesting that chloroquine might avoid further lysosomal degradation and facilitate the release of endosome-trapped cytC-gal into cytosol to exert therapeutic action. However, in the other cell line, MCF7, co-incubation of chloroquine had no further effect on cellular viability, suggesting that cytC was uptaken via endosome-independent pathway in MCF cells. Although recombinant arginine deiminase (rADI) is a potent cell proliferation inhibitor towards most hepatocellular carcinoma and melanoma, rADI resistance is still observed in certain tumor cells, such as HepG2. Our lab previously showed intracellular delivery of rADI into resistant cells by cell-penetrating peptide restored sensitivity to rADI treatment. For this reason, we conjugated galactose-CHO to rADI in order to restore rADI sensitivity on HepG2 cells. We also performed cell viability assay and results shown that galactose modification could significantly inhibit cell proliferation, implying more rADI delivered into HepG2 cells. To conclude, galactose-CHO could conjugate galactose ligand to both cytC and rADI protein and enhance anti-proliferation effect in HepG2 and MCF7 cells. The cellular uptake into HepG2 cells were via ASGPR; the cellular uptake for MCF7 cells was not observed, therefore the reason of anti-proliferation effect on MCF7 cells remained unclear. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T17:52:39Z (GMT). No. of bitstreams: 1 ntu-101-R99423022-1.pdf: 940316 bytes, checksum: dd454ebf7e9cf79d260fbdb751443224 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | 目錄 I
圖目錄 IV 縮寫表 V 中文摘要 VI Abstract IX 第一章 緒論 1 1.1 蛋白質藥物的發展與胞內運輸 1 1.2 主動標靶遞送 (Active Targeting Delivery) 3 1.3 ASGPR (asialoglycoprotein receptor) 4 1.4 精胺酸 (arginine) 與基因重組精胺酸去亞胺酶 (recombinant arginine deiminase,rADI) 4 1.5 將糖基鍵結至蛋白質之方法 – 還原胺化反應 (reductive amination) 6 1.6 細胞色素 C (cytochrome c;cytC) 6 第二章 實驗目的 8 第三章 實驗材料 10 3.1. 細胞培養 10 3.2. 還原胺化反應 10 3.3. 蛋白質濃度測定 11 3.4. 一級胺定量 11 3.5. 細胞存活分析 11 3.6. 細胞色素C之酵素活性測定 11 3.7. 螢光素標記 11 3.8. 製備基因重組精胺酸去亞胺酶 (rADI) 12 第四章 實驗方法 14 4.1. 細胞培養 (cell culture) 14 4.2. Galactose-CHO 合成及純度確認 14 4.3. 還原胺化反應 (reductive amination) 15 4.4. 蛋白濃度測定 (BCA assay) 16 4.5. 一級胺定量 (primary amine quantification) 16 4.6. 半乳糖糖基數估算 17 4.7. 螢光素標記法 (fluorescein labeling method) 17 4.8. 細胞攝取試驗 (cellular uptake assay) 18 4.9. 細胞存活率試驗 (cell viability assay) 19 4.10. CytC 之酵素活性測定 20 4.11. rADI 的胞內遞送 20 4.11.1 rADI 的製備 20 4.11.2 rADI 的活性測定 (activity assay) 21 4.12. 統計分析 21 第五章 實驗結果 22 5.1. 利用galactose-CHO 可鍵結半乳糖至蛋白質 22 5.2. 鍵結半乳糖基對於 cytC 的細胞攝取量之影響 22 5.3. 鍵結半乳糖基可將 cytC 遞送至細胞內產生細胞毒性 24 5.4. 鍵結半乳糖基的數目對cytC的酵素活性之影響 25 5.5. 投予相同酵素活性的 cytC-gal 對細胞存活率之影響 26 5.6. 添加 endosomal escape agent (chloroquine) 對細胞存活率之影響 27 5.7. 鍵結半乳糖基對 rADI 酵素活性之影響 27 5.8. 鍵結半乳糖基增加 rADI 的細胞毒性 28 第六章 討論 29 6.1 Galactose-CHO 可縮短鍵結半乳糖至蛋白質的反應時間 29 6.2 經由半乳糖基修飾後的 cytC 在 HepG2 及 MCF7 細胞株的不同細胞攝取機制 30 6.3 鍵結半乳糖基降低 cytC 及 rADI 之酵素活性 31 6.4 半乳糖基是否能專一性地標靶治肝臟的 ASGPR 33 6.5 鍵結半乳糖基的數目與細胞攝取量的關聯性 34 6.6 藉由 chloroquine 的共同培養判斷細胞攝取是否為 endosome - mediated 35 6.7 鍵結半乳糖基可增加 rADI 的胞內遞送 36 6.8 實驗限制 37 第七章 結論 38 第八章 參考文獻 58 | |
dc.language.iso | zh-TW | |
dc.title | 蛋白質與半乳糖接合體在不同癌細胞株的胞內遞送效果 | zh_TW |
dc.title | Intracellular delivery of protein and galactose conjugates in cancer cell lines | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 林文貞,邱士娟 | |
dc.subject.keyword | 半乳糖,主動標靶,ASGPR,蛋白質遞送,基因重組精胺酸去亞胺酶, | zh_TW |
dc.subject.keyword | galactose,active targeting,ASGPR,protein delivery,recombinant arginine deiminase, | en |
dc.relation.page | 69 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-13 | |
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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