利用聚乙烯亞胺輸送環狀核苷酸磷酸二酯酶4B之專一性反義寡核苷酸可抑制LPS刺激小鼠巨噬細胞釋放TNF-α;Delivery of antisense oligonucleotides specific to phosphodiesterase 4B gene by polyethylenimine downregulates LPS-induced TNF-α release in mouse macrophages

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題名:	利用聚乙烯亞胺輸送環狀核苷酸磷酸二酯酶4B之專一性反義寡核苷酸可抑制LPS刺激小鼠巨噬細胞釋放TNF-α;Delivery of antisense oligonucleotides specific to phosphodiesterase 4B gene by polyethylenimine downregulates LPS-induced TNF-α release in mouse macrophages
作者:	李太榮;Lee,Tai-rong
貢獻者:	生命科學系
關鍵詞:	環狀核苷酸磷酸二酯酶4B;聚乙烯亞胺;反義寡核苷酸;腫瘤壞死因子
日期:	2016-01-28
上傳時間:	2016-03-17 19:19:40 (UTC+8)
出版者:	國立中央大學
摘要:	PDE4B在免疫細胞內會參與多種免疫發炎反應，且與許多發炎疾病息息相關，因此若能有效調控PDE4B mRNA表現及PDE4B酵素生成應可有效減緩該等發炎反應之相關病症。已知PDE4抑制劑roflumilast與apremilast為有效的抗發炎藥劑，但在臨床上會產生副作用如噁心、腸炎等使療效受限，而研究顯示該等副作用主要是由於PDE4抑制劑缺乏PDE4B選擇性所致，因此，研發PDE4B專一性藥劑將有助於發炎疾病的治療。有鑑於此，本研究擬以PDE4B專一性反義寡核苷酸(Antisense oligonucleotide, ASO)藉由反義寡核苷酸運輸策略(antisense approach)達到此目的。已知DNA分子本身無法有效穿越細胞膜進入細胞內，因此需藉由不同的載體輸送，如脂質體(liposome)、穿膜胜肽(Cell-penetrating peptide)或正電荷聚合物(cationic polymer)等。本研究主要是利用正電荷聚合物-聚乙烯亞胺(Polyethylenimine, PEI)為載體以檢測其是否可有效輸送ASO到免疫細胞內調控免疫發炎反應。我們將PEI分別與不同的TNF-α和PDE4B ASO形成複合物，並轉染至Raw 264.7巨噬細胞以評估其調控LPS誘導TNF-α產生的功效。結果顯示，以不同的PEI與ASO電荷比(N/P ratio)進行實驗，只有N/P為100時會抑制TNF-α的釋放，而在四個TNF ASO (TNF1 ~ 4 ASO)中，僅PEI/TNF3 ASO複合物有顯著抑制作用，且其抑制程度(約30%)與控制組TNF5 ASO (約33%)相當。再者，將PEI與四個PDE4B ASO (PDE4B-1 ~ 4 ASO)形成複合物進行實驗，我們發現PDE4B-2、PDE4B-3與PDE4B-4 ASO均可顯著抑制LPS誘導TNF-α的釋放且無明顯的細胞毒性，其中又以PDE4B-4 ASO效果最佳(抑制約58%)。此外，PDE4B-3與PDE4B-4 ASO之抑制程度均較TNF ASO直接抑制TNF-α mRNA為佳。由這些結果推測，PDE4B ASO是經由抑制細胞內PDE4B mRNA，進而降低PDE4B酵素生成，使cAMP濃度上升並活化PKA，再間接抑制LPS誘導TNF-α的產生。我們進一步以動態雷射散射粒徑分析儀檢測PEI/ASO複合物的特性，結果顯示，於N/P電荷比為100時，其表面電位介於30 ~ 45 mV，且粒徑大小約為400 nm ~ 600 nm，此符合胞吞作用進行的條件。綜合以上結果，我們認為PEI/PDE4B-4 ASO於N/P電荷比為100時，會有效進入巨噬細胞內並抑制發炎反應，且又無明顯的細胞毒性，因此以PEI輸送PDE4B-4 ASO極具潛力應用於基因治療上。;Phosphodiesterase 4B (PDE4B) is essential for various immune and inflammatory responses and hence plays important role in the pathogenesis of several inflammatory diseases. The nonselective PDE4 inhibitors, including the clinically approved roflumilast and apremilast, have been shown to effectively ameliorate inflammatory diseases, but the side effects such as emesis and enteritis have limited their dosing and thereby the immunomodulatory effects. Therefore, development of inhibitors selective for PDE4B is considered a promising approach to improve the therapeutic windows. To this purpose, different PDE4B-specific antisense oligonucleotides (PDE4B ASO) were designed in this study and the cationic polymer polyethylenimine (PEI) was employed as a carrier to deliver the ASO into Raw 264.7 macrophages to test their effect on lipopolysaccharide (LPS)-stimulated TNF-α production. The TNFα-specific ASOs were also designed for comparison. The results showed that at the ASO concentration of 0.2 uM and N/P charge ratio of 100, several ASO tested significantly suppressed LPS-induced TNF-α release without cytotoxicity. Among the four TNF-α ASO (TNF1~4 ASO) tested, only TNF3 ASO produced inhibitory effect on TNF-α release, and its level of inhibition was similar to that of the control TNF5 ASO. As for the four PDE4B ASO (PDE4B-1~4 ASO), three (PDE4B-2, 3, and 4) were shown to significantly inhibit the LPS-induced TNF-α release, and among which the PDE4B-4 ASO displayed the highest inhibition (approximately 58%). Moreover, the PEI/PDE4B-3 and PEI/PDE4B-4 complexes were more effective in blocking the TNF-α release compared to all PEI/TNF-α ASO tested. The analysis of PEI/ASO complexes by dynamic laser scattering (DLS) revealed that at the N/P ratio of 100 all of the complexes had the surface charge of 30-45 mV and the size of 400-600 nm, which is known to be appropriate for endocytosis of nanoparticles. Collectively, the results indicated that the PEI/PDE4B-4 ASO complex at the ASO concentration of 0.2 uM and N/P ration of 100 may reproduce the anti-inflammatory effects of nonselective PDE4 inhibitors but devoid of their side effects. Therefore, it has the potential for therapeutic application in inflammatory diseases.
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