自我吞噬產生會保護HeLa細胞免於PEI和PLL所誘導之細胞死亡

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Title:	自我吞噬產生會保護HeLa細胞免於PEI和PLL所誘導之細胞死亡 Autophagy protects HeLa cells against polyethylenimine- and poly(L-lysine)-induced cell death
Authors:	林嘉緯
Contributors:	嘉南藥理科技大學：藥物科技研究所郭榮華
Keywords:	自我吞噬細胞凋亡細胞壞死 autophagy apoptosis necrosis
Date:	2011
Issue Date:	2011-10-27 11:23:44 (UTC+8)
Abstract:	陽離子載體 PEI、PLL 聚合物被當作為非病毒性載體來傳遞核酸，可應用在基因治療。然而兩種聚合物有明確的細胞毒性，但確切的分子機轉仍未清楚。本論文中，我們證明了 PEI 和 PLL 會誘導人類子宮頸癌細胞 (HeLa) 產生細胞自我吞噬之情形：從細胞形態上 PEI 和 PLL 會造成HeLa 細胞質中形成半透明之空泡。PEI 劑量在 20 和 40 g/mL 於有加入溶酶體蛋白酶抑制劑 (E64-d and Pepstatin A) 或未加入溶酶體蛋白酶抑制劑都會有 LC3-II 的累積，PLL 劑量在 40 g/mL 加入溶酶體蛋白酶抑制劑有 LC3-II 的累積，LC3-I 轉變成 LC3-II 為細胞自噬之主要指標，當細胞培養於含 PEI (或PLL) 與 3-MA 2、4 小時後，可以明顯看到較未加入 3-MA，僅PEI或PLL所引發之細胞毒性更高，我們實驗得到結論為 PEI 和 PLL 在人類子宮頸癌細胞中會誘導自我吞噬與細胞凋亡， PEI 有逃脫 endosomal 的能力，在沒有加入溶酶體蛋白酶抑制劑就有自我吞噬流通量的變化，本實驗能更深入了解到陽離子聚合物 PEI 和 PLL 誘導之細胞死亡機轉。 Polyethylenimine (PEI) and poly(L-lysine) (PLL)，cationic polymer vectors used for gene therapy，are known to be cytotoxic，but their molecular mechanisms of cell death are not fully understood. We provide evidence that PEI and PLL induced autophagy in HeLa cervical cancer cells. Morphologically，PEI- and PLL-treated cells had more regular translucent vacuoles in the cytoplasm. Conversions of the autophagosomal markers LC3-I to LC3-II were significant，respectively，in cells treated with both 20 and 40 g/mL of PEI (with or without the lysosomal protease inhibitors E64-d and Pepstatin A)，and in cells treated with 40 g/mL of PLL in the presence of E64-d and Pepstatin A. Cell death was higher in cells treated for 2 and 4 h with PEI plus autophagy inhibitor 3-methyladenine (3-MA) and with PLL plus 3-MA. We conclude that，in contrast to apoptosis，autophagy protected PEI- and PLL-treated HeLa cancer cells from cell death. Also，PEI’s ability of endosomal escape facilitates the detection of autophagic flux without using lysosomal protease inhibitors. Our study may provide a deeper insight into the molecular mechanisms of cell death caused by cationic polymers.
Relation:	校內校外均不公開，學年度：99，77頁
Appears in Collections:	[Dept. of Pharmacy] Dissertations and Theses

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