人類巨噬細胞U937和陽離子樹狀體之細胞交互作用

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Title:	人類巨噬細胞U937和陽離子樹狀體之細胞交互作用 Interactions between U-937 human macrophages and cationic dendrimers
Authors:	林宜陵 Yi-Lin Lin
Contributors:	郭榮華嘉南藥理科技大學：藥物科技研究所
Keywords:	巨噬細胞陽離子樹狀體反應性氧分子粒腺體膜電位細胞週期流式細胞儀 RNA純化 DNA/dendrimer複合體膠體電泳 Macrophages Cationic dendrimer Reactive oxygen
Date:	2008
Issue Date:	2010-06-01 09:21:32 (UTC+8)
Abstract:	本實驗利用PPI樹狀體和人類巨噬細胞株 (U937) 觀察其之間的交互作用反應：其中我們又以反應性氧分子 (reactive oxygen species ; ROS) 含量，粒腺體膜電位的變化，細胞大小、胞器複雜度改變以及細胞週期進行探討。在此所使用的是PPI樹狀體合成代數第二代 (generation 2) DAB 2.0和第三代 (generation 3) DAB 3.0。結果顯示ROS的變化，DAB 2.0 並沒有在隨著時間與劑量增加的培養週期中，使H2O2產生一致的改變；另外，DAB 2.0 使細胞在培養週期中產生的O2-除了十二小時外，其餘則是趨於上升的變化。比較於DAB 3.0，在所有培養週期中任何時間與任何劑量上都沒有H2O2的產生；而O2-則是於六小時的60 g/mL微微增加外，其餘時間及劑量上也沒有明顯變化。在這些結果中顯示巨噬細胞內所產生的反應性氧分子深深受到樹狀體表面所帶的電荷所影響，而產生不穩定的變動情形。DAB 2.0加到巨噬細胞培養時，除了三小時外，粒腺體的細胞膜電位在所有培養週期及所有劑量中皆呈現增加；相對的，DAB 3.0與其他培養週期的時間相比，在六小時則出現了明顯的波動現象。另外，當巨噬細胞曝露於PPI樹狀體時，可觀察到細胞大小及胞器明顯的改變，尤其是DAB 3.0所引起的細胞反應又比DAB 2.0明顯釵h。在細胞週期方面，我們利用 propidium iodide 螢光染劑染上DNA，除了一小時的DAB 2.0 和DAB 3.0短時間培養外，其餘培養時間的細胞週期比起未加入樹狀體培養的控制組細胞都出現細胞凋亡情形產生。PPI樹狀體所引起巨噬細胞的反應性氧分子的產生及粒腺體膜電位的變化，皆與其他研究者所報導的基因載體例如一些帶正電的脂質體 (cationic liposomes) 和 polyalkylcyanoacrylate 所造成的反應有很大的不同。而大多數的研究者，只在於探討這些基因傳輸載體對於標的物目標的傳送能力，卻很少有研究者探討這些電荷間非專一性交互作用。因此我們將針對這些非專一性的正電荷樹狀體模擬在傳輸後所殘留的正電荷與細胞內RNA之交互作用所進行研究。首先，先將人類巨噬細胞培養於 PAMAM、 PPI樹狀體及 DNA/dendrimer 複合體中，再利用常見將細胞打碎 (monophasic lysis) 的方式來分離出 RNA，並進行電泳分析。為了評估不同種類的細胞間是否會有不同的情形產生，我們也選用了老鼠纖維母細胞 (NIH/3T3) 來進行相同的實驗。經由實驗證實， PAMAM和PPI樹狀體所帶的正電荷確實會影響到RNA的膠體電泳泳動能力。在此，我們發現了這些帶正電的樹狀體對於巨噬細胞間的交互作用一個新的觀點，對於未來使用這些樹狀體於分子層面的應用上，更應該考量到細胞毒性及其引發的免疫反應。並且在於RNA純化的過程及應用上，要盡量避免這些帶正電荷樹狀體的干擾作用。 Interest in using poly(propyleneimine) (PPI) dendrimers and Cationic dendrimers such as poly(amidoamine) (PAMAM) for biomedical applications is increasing. Before using dendrimers in vivo, their interactions with macrophages must be fully understood because they are primarily removed from circulation by the macrophages of the mononuclear phagocyte system. However, few investigators have studied in detail the intracellular responses that cationic dendrimers induce in macrophages. Here we examined the intracellular responses—reactive oxygen species (ROS) content, mitochondria membrane potential, cell size and complexity, and cell cycle profiles—in U-937 human macrophages treated with poly(propyleneimine) dendrimers generation 2 (DAB 2.0) and 3 (DAB 3.0). Our study focused on the concentration ranges within which cell viability was greater than 90% after PPI dendrimers had been incubated for 16 h. For spontaneous ROS generation, DAB 2.0 did not consistently generate hydrogen peroxide production with increasing dosages over the entire culture period while it was capable of generating superoxide content except during the 12 h of incubation. In contrast, DAB 3.0 did not induce any hydrogen peroxide and superoxide production except for an abrupt increase of superoxide content at 60 μg/mL after 6 h of incubation. Our results showed that ROS responses in macrophages were strongly influenced by the nature of the dendrimer surface. Except at 3 h, DAB 2.0 increased mitochondrial membrane potential for every dose and culture period. In contrast, DAB 3.0 caused a significant fluctuation in mitochondrial membrane potential only at 6 h, compared with other incubation times. Exposing macrophages to PPI dendrimers caused dramatic and significant changes in macrophage cell size and complexity, and DAB 3.0 caused greater changes than DAB 2.0 did. For incubation times longer than 1 h, propidium iodide staining showed that cells treated with DAB 2.0 and 3.0 had a higher subG1 phase (indicative of apoptosis) than did untreated cells. PPI dendrimers induced different activated patterns in ROS generation and changes of mitochondrial membrane potential than did other carriers such as cationic liposomes and polyalkylcyanoacrylate. Also, very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that poly(amidoamine) (PAMAM) and PPI dendrimers strongly altered the mobility of RNA in the gels. Our findings provide a novel insight into the cytotoxic effects at the molecular level that dendrimers cause in macrophages and such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.
Relation:	校內外完全公開
Appears in Collections:	[Dept. of Pharmacy] Dissertations and Theses

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