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研究生:邱鉉文
研究生(外文):Hsuan-Wen Chiu
論文名稱:陰陽離子性液胞和陽離子樹枝體對RAW264.7老鼠巨噬細胞株死亡機轉探討
論文名稱(外文):Cytotoxicity characterization of catanionic vesicles and cationic dendrimers in RAW 264.7 murine macrophage-like cells
指導教授:郭榮華郭榮華引用關係
指導教授(外文):Jung-Hua Kuo
學位類別:碩士
校院名稱:嘉南藥理科技大學
系所名稱:生物科技系暨研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:70
中文關鍵詞:細胞凋亡陰陽離子性液胞陽離子樹枝體
外文關鍵詞:Catanionic vesiclesCationic dendrimerApoptosis
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陰陽離子性液胞 (catanionic vesicles),由於界面活性劑的來源豐富,且價格較磷脂質廉價,製備簡單,以及較磷脂質安定,因此陰陽離子界面活性劑具有成為新型非病毒性載體及未來在疫苗佐劑方面之應用的潛力。另外陽離子樹枝體 (catanionic dendrimers) 其具有奈米的大小之特性,使其常用於藥物或基因的輸送系統。目前有關陰陽離子性液胞和陽離子樹枝體對細胞細胞死亡機轉的探討文獻是比較少的。
這些成分對於生物相容性以及未來應用人體應用時的安全性是非常重要,所以對相關毒性測試及運用範圍的瞭解是不可缺的。在本實驗中我們探討為由陰離子型界面活性劑 (Ploypropylenimine;SDS) 和陽離子型界面活性劑 (Hexadecyltrimethylammonium bromide; HTAMB) 以及膽固醇所組成的陰陽離子性液胞,造成老鼠巨噬細胞株死亡,及可能的死亡機轉做一個探討。陰陽離子性液胞的添加劑量和細胞的存活率有呈現典型 dose-dependent 的對應關係,由螢光顯微鏡下觀察染色質濃縮, DNA 的電泳呈現片段化,以及在 propidium iodide 染色與流式細胞儀的分析結果顯示,Sub-G1 分佈的亞二倍體 (hypodiploid DNA),隨著陰陽離子性液胞劑量的增加而增加證明。陰陽離子性液胞會誘發細胞凋亡。此外廣泛性 caspase inhibitor (zVAD-fmk),無法抑制陰陽離子性液胞所引起之細胞凋亡,因此我們推斷陰陽離子性液胞所造成細胞凋亡的路徑為 caspase-independent pathway。由上述結論對於陰陽離子性液胞對細胞所誘導的死亡機轉提出一個新的詮釋

陽離子樹枝體對老鼠巨噬細胞株的毒性測試,我們選擇一般較為常用的 Polyamidoamine (PAMAM) 與 ploypropylenimine (DAB) 兩種陽離子樹枝體作為研究材料,陽離子樹枝體添加劑量和細胞的存活率同樣呈現典型 dose-dependent 的現象,隨劑量越高,細胞存活率越低,且也會誘發細胞凋亡的產生。流式細胞儀結果分析顯示,凋亡細胞的數量隨著陽離子樹枝體劑量的增加而增加。且 DAB 相較於相同濃度劑量的 PAMAM 所誘發細胞凋亡的程度較為嚴重,此外與 caspase inhibitor (zVAD-fmk) 共同處理細胞。結果顯示陽離子樹枝體所造成細胞凋亡的路徑可能為 caspase-dependent pathway。

我們同時也觀察陰陽離子性液胞和陽離子樹枝體對於其他包括老鼠母纖維細胞株 (NIH/3T3) 和老鼠肝細胞株 (BNL CL.2),是否也會造成凋亡,然而結果顯示並沒有發生細胞凋亡的現象。本研究的結果顯示陰陽離子液胞和陽離子樹枝體皆會誘導老鼠巨噬細胞細胞凋亡的發生,這些結果可做為日後發展藥物或基因的輸送系統時可做為生物可相容性的重要參考資訊。
Catanionic vesicles possess more attractive properties such as stability and lower cost, and these characteristics may make them suitable as a non-viral vehicle and for other biomedical applications such as vaccine adjuvants. Also, cationic dendrimers possess attractive nano-sized architectures and these characteristics make them suitable as targeted drug/gene delivery systems. However, very little is known about their possible cytotoxic mechanisms in cellular system.
Also, this information is vital for the future development of safe biomedical systems. In the current study, the cytotoxic effect of catanionic vesicles, consisting of anionic surfactant (SDS), cationic surfactant (HTMAB), and cholesterol, in cultured RAW 264.7 murine macrophage-like cells was determined. The treatment of catanionic vesicles produced a dose-dependent effect on macrophage cells. RAW 264.7 cells exposed to catanionic vesicles exhibited morphological features of apoptosis such as chromatin condensation. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated by catanionic vesicles. Analysis from flow cytometry demonstrated an increase of hypodiploid DNA population (sub-G1) and a simultaneous decrease of diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells with catanionic vesicles. In addition, it was shown that pretreatment of RAW 264.7 cells with the general caspase inhibitor (zVAD-fmk) did not prevent apoptosis induced by catanionic vesicles, suggesting that apoptosis in macrophage cells followed a caspase-independent pathway induced by catanionic vesicles. These data provide novel insight into the effect of catanionic vesicles on the mechanisms of cell death induced by catanionic vesicles.
For cationic dendrimers, the apoptotic and necrotic effects of starburst polyamidoamine (PAMAM) and polypropylenimine (DAB) dendrimers in cultured RAW 264.7 murine macrophage-like cells were investigeted. Cationic dendrimer treatment produced a typically dose-dependent cytotoxic effect on macrophage cells. RAW 264.7 cells exposed to cationic dendrimers exhibited morphological features of apoptosis. Apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated by cationic dendrimers. Analysis from flow cytometry demonstrated an increase of hypodiploid DNA population (sub-G1) and a simultaneous decrease of diploid DNA content, indicating that DNA cleavage occurred after exposure of the cells with cationic dendrimers.
Also, cells treated with DAB dendrimer induced a higher percentage of sub-G1 population than those treated with PAMAM dendrimer at the same dose. In addition, it was shown that pretreatment of RAW 264.7 cells with the general caspase inhibitor (zVAD-fmk) did prevent some degree of apoptosis induced by cationic dendrimers, suggesting that apoptosis in macrophage cells involved a caspase-dependent pathway. We also found that macrophage cells were sensitive to induction of apoptosis by dendrimers, and catanionic vesicles, whereas NIH/3T3 cells (mouse fibroblast) and BNL CL.2 (mouse liver) cells did not undergo apoptosis. These data could be helpful for optimizing the biocompatibility of dendrimers used for targeted drug/gene delivery.
中文摘…………………………………………………………………Ι
英文摘要………………………………………………………………Ⅵ
誌謝……………………………………………………………………Ⅶ
目錄……………………………………………………………………Ⅷ
表目錄 ………………………………………………………………ⅩⅠ
圖目錄 ………………………………………………………………ⅩⅠ
縮寫表 ………………………………………………………………ⅩⅣ

第一章 緒論…………………………………………………………1
1-1、基因治療(gene therapy)………………………………………1
1-1-2、基因遞送系統…………………………………………………2
1-1-2-1、病毒性基因載體……………………………………………2
1-1-2-2、非病毒性基因載體…………………………………………2
1-1-2-3、物理性基因轉染……………………………………………2
1-1-2-4、化學方式的基因轉染………………………………………4
1-1-2-5、為何考量使用非病毒性基因載體…………………………8
1-2、細胞凋亡(Apoptosis) ………………………………………9
1-2-1、細胞凋亡的機轉………………………………………………10
1-2-2、caspase-dependent 途徑而引起細胞凋……………………10
1-2-3、caspase-independent 途徑而引起細胞凋亡………………12
1-3、研究動機與目的…………………………………………………14

第二章 材料與方法……………………………………………………15
2-1、化學藥品及試劑…………………………………………………15
2-2、實驗儀器…………………………………………………………16
2-3、實驗製備與方法…………………………………………………17
2-3-1、陰陽離子液胞的製備…………………………………………17
2-3-1-1、離子對雙親分子 (ion pair amphiphilie(IPAs)) 的製備
………………………………………………………………17
2-3-1-2、帶電陰陽離子液胞 (Catanionic vesicles) 的製備 …18
2-3-2、粒徑分布………………………………………………………19
2-3-3、界面電位………………………………………………………20
2-4、細胞的培養………………………………………………………20
2-4-1、細胞株繼代培養( subculture )方法………………………20
2-4-2、細胞存活率 (cell viability) 之測定……………………22
2-4-3、乳糖去氫酶釋放之測定 Lactate dehydrogenase (LDH)
release assay…………………………………………………23
2-4-4、細胞型態觀察…………………………………………………23
2-4-5、細胞凋亡 (Apoptosis) 之型態觀察 ………………………24
2-4-6、凋亡偵測………………………………………………………24
2-4-6-1、DNA片段化分析 (DNA fragmentation assays)…………24
2-4-7、以流式細胞儀做細胞週期做細胞凋亡的分析………………27
2-4-7-1、DNA content ………………………………………………28
2-4-7-2、zVAD-fmk inhibition ……………………………………28
2-5、統計分析…………………………………………………………31
第三章 結果……………………………………………………………32
3-1、陰陽離子性液胞(Catanionic vesicles)對於老鼠巨噬細胞
RAW264.7 之影響 ………………………………………………32
3-1-1、陰陽離子性液胞物理安定性…………………………………32
3-1-2、對細胞存活率 (cell viability) 之影響…………………32
3-1-3、對細胞細胞膜的完整性之影響………………………………33
3-1-4、對於細胞型態 (cell morphology) 之影響 ………………33
3-1-5、DNA 片段化分析(DNA fragmentation assays)……………34
3-1-6、對於細胞週期之影響…………………………………………34
3-2、陽離子樹枝體(Cationic dendrimers) : 對於老鼠巨噬細胞
RAW264.7之影響…………………………………………………36
3-2-1、對細胞存活率 (cell viability) 和細胞細胞膜的完整性
之影響…………………………………………………………36
3-2-2、對於細胞型態 (cell morphology) 之影響 ………………36
3-2-3、DNA 片段化分析 (DNA fragmentation assays) …………37
3-2-4、對於細胞週期之影響…………………………………………38

第四章 討論……………………………………………………………39
4-1、陰陽離子液胞 (catanionic vesicles)……………………39
4-2、陽離子樹枝體 (Cationic dendrimers)……………………43

第五章 結論……………………………………………………………47
參考文獻 ………………………………………………………………48


表目錄
1.表(一)添加陽離子樹枝體後細胞週期的分佈(cell cycle)的情
形 ……………………………………………………69

圖目錄
1.(附一) 基因治療(gene therapy)簡圖……………………………1
2.(附二) 陰陽離子性液胞的組成簡圖………………………………6
3.(附三) 細胞凋亡途徑簡圖…………………………………………12
4.(附四) 陰陽離子液胞 (Catanionic vesicles) 的製備簡圖 …19
5.(附五) Caspase 抑制劑所抑制路徑之簡圖………………………29
6.圖(一)陰陽離子性液胞的(A)界面電位與(B)粒徑分佈 ………58
7.圖(二)陰陽離子性液胞在不同濃度下處理後,細胞存活率與LDH
釋放的含量 …………………………………………………………59
8.圖(三)未經陰陽離子性液胞處理和陰陽離子性液胞的處理 48小
時後,RAW 264.7細胞株型態圖……………………………………60
9.圖(四)未經陰陽離子性液胞處理和陰陽離子性液胞的處理 48小
時後利用PI來染細胞的細胞核 (PI nuclear staining) 觀察細胞
凋亡的特徵 …………………………………………………………61
10.圖(五)針對不同濃度的陰陽離子性液胞所做的DNA 片段化的電
泳分析圖……………………………………………………………62
11.圖(六)添加陰陽離子性液胞後細胞週期的分佈 (cell cycle)
的情形………………………………………………………………63
12.圖(七)有未經過Inhibitor處理過再添加陰陽離子性液胞和有
經過Inhibitor處理過再添加陰陽離子性液胞細胞週期的分佈
(cell cycle) 情形 ………………………………………………64
13.圖(八)陽離子樹枝體對在不同濃度下處理後,細胞存活率與
LDH釋放的含量 ……………………………………………………65
14.圖(九)未經陽離子樹枝體處理和陽離子樹枝體處理 24小時後
,RAW 264.7細胞株型態圖……………………………………………66
15.圖(十)未經陽離子樹枝體處理和陽離子樹枝體處理 24小時後
利用PI來染細胞的細胞核(PI nuclear staining) 觀察細胞凋亡
的特徵 ………………………………………………………………67
16.圖(十一)針對不同濃度的陽離子樹枝體所做的DNA 片段化的電
泳分析圖 ……………………………………………………………68

自述 ……………………………………………………………………70
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