研究不同酚類化合物對肝纖維化星狀細胞生長之調控與治療：利用玻尿酸-聚乳酸奈米粒子包覆薑黃素之藥物傳遞

Regulation of activated hepatic stellate cytotoxicity by polyphenolic compounds and their targeting delivery using hyaluronic acid-polylactic acid nanoparticles

劉怡婷(Yi-Ting Liu)

指導教授 ： 吳立真

國立暨南國際大學/科技學院/生物醫學科技研究所/碩士(2011年)

摘要

本研究主要探討酚類化合物對活化之肝星狀細胞生長的調控，並藉由奈米粒子作為藥物傳遞載體以提升纖維化治療之效率為目的。論文共分三部分進行研究: (1) 黃酮類化合物-槲皮素 (Quercetin)是否有效抑制活化之肝星狀細胞增殖、(2) 酚酸類化合物-沒食子酸 (Gallic acid)如何誘導肝星狀細胞死亡以減緩肝纖維化表徵、(3) 以玻尿酸-聚乳酸奈米粒子包覆疏水性酚類化合物(薑黃素)應用於肝纖維化治療之可行性。
研究中將細胞分別與槲皮素 (Quercetin)、沒食子酸 (Gallic acid)共培養24小時後，偵測細胞存活率 (MTT assay)、DNA合成速率分析 (BrdU assay)，證實槲皮素與沒食子酸能有效抑制活化之肝星狀細胞生長;在細胞週期分析中也發現槲皮素與沒食子酸會使細胞週期停滯於G1期，且透過Fas/Fas ligand pathway誘導細胞走向凋亡。此外，在基因微陣列系統分析中除了證實沒食子酸具抗氧化能力外，也發現沒食子酸可能藉由增加氧化壓力，促使胞內鈣離子濃度過高，進而活化Calpain、引發細胞凋亡，最終使細胞膜受損、脹破、細胞死亡。活化的肝星狀細胞在肝纖維化過程中扮演重要角色，因此我們進行膠原蛋白定量與纖維化標的蛋白α-SMA (α-smooth muscle actin)表現量分析，證實沒食子酸可減緩肝纖維化表徵，有利於肝纖維化治療。
傳統的給藥系統大多是透過口服、注射或塗抹方式，唯有藥物進入血液才能循環至患部，故藥效取決於血液中藥物濃度，然而藥物半衰期短，加上人體新陳代謝作用，需多次給藥才能維持送入細胞內作用的藥物濃度，以提升肝纖維化治療效率。本研究利用HA-PADH-PLA 為藥物載體，傳送酚類化合物-薑黃素到活化之肝星狀細胞。實驗包含玻尿酸奈米粒子的製備，以粒徑儀、掃描式電子顯微鏡 (SEM)等確定粒子形成顆粒以及大小後，包埋螢光物質 (Coumarin 6)或抗纖維化藥物 (Curcumin)對肝臟組織細胞進行體外測試 (in vitro)。從實驗中證實HA-ADH-PLA polymer對細胞毒性甚小，可視為無毒性 ; 且以玻尿酸奈米粒子作為載體後，可降低薑黃素對活化的肝星狀細胞之半致死濃度 (IC50)降低30倍之多 (由 80.4 μM降低至 2.6 μM) ; 以 CD44 抗體進行定量分析，證實活化的肝星狀細胞其表面 CD44 較未活化的肝星狀細胞多; 於靶向試驗中也發現到隨著肝星狀細胞活化程度的不同，細胞所攝入之螢光玻尿酸奈米粒持在有效範圍。因此本論文第三部分作以下假設:若可以使用生物可降解的高分子-玻尿酸 (HA)包埋對肝纖維化具治癒效果的薑黃素 (Curcumin)，利用肝竇中活化的肝星狀細胞表面有大量之玻尿酸接受器 CD44 會和 HA 結合的機轉，將薑黃素子亦具有顯著差異。
綜合以上研究結果可知，多酚類化合物-槲皮素、沒食子酸抑制活化之肝星狀細胞生長，並透過Fas/Fas ligand pathway或Intracellular Calcium signaling誘導細胞走向死亡。本研究更發展出新穎之藥物載體 HA-ADH-PLA奈米粒子，藉由HA對CD44具靶向作用，增加疏水性藥物於身體可用率，以提升纖維化治療之效率。

關鍵字

槲皮素 ； 沒食子酸 ； 玻尿酸 ； 聚乳酸 ； 奈米粒子 ； CD44 ； 肝纖維化

並列摘要

Hepatic stellate cells (HSCs), a central role of fibrosis process, transform from quiescent types to activated (aHSCs) ones, concomitantly with the production of significant amount of ECM. Significant aHSC apoptosis engaged can be observed after the recovery of acute hepatitis. Thus the eradication of aHSC potentially becomes a mean for hepatofibrosis treatment. Herein phenolic compounds (quercetin, gallic acid) were evaluated to investigate their effect on the proliferation and apoptosis of aHSCs. Meanwhile phenolic compound (curcumin) encapsulated nanoformulation by HA-PLA nanoparticle drug delivery system was established to enhance the efficacy on aHSC eradication. Treatment of aHSCs with quercetin and gallic acid inhibited cell viability in a dose- and time-dependent manner. Results revealed that increased BrdU incorporation suggested quercetin and gallic acid restrained aHSC proliferation. Additionally, quercetin limited aHSC proliferation by inducing a G1 arrest as evidenced by decreased expression of cyclin D1、D2、A、B1、E. Moreover quercetin and gallic acid induced aHSC apoptosis via Fas/Fas ligand-mediated extrinsic pathway. Gallic acid also decreased SMA expression as well as collagen production, and caused cell death by calcium signaling-induced ER stress. Gene microarray analysis showed that gallic acid induced several regulations on aHSC including extrinsic apoptosis as evidenced by the increase of Fas/Fas ligand expression, promotion of anti-fibrotic and anti-oxidative gene expression. For nanoformulation, the physicochemical properties such as size and shape of HA-PLA nanoparticles were characterized by particle size analyzer and scanning electron microscopy (SEM). Fluorescent dye (coumarin-6) and curcumin-encapsulated nanoparticles were treated with cancer cell line or HSCs (qHSC and aHSC) in vitro. Nanoparticle cellular binding efficiency was examined on several cell types such as MDA-MB-231 (CD44+), ZR-75-1 (CD44-), A549, aHSCs and qHSCs. High fluorescent intensity was shown in aHSCs due to the elevated expression of CD44, the receptor of HA, on aHSCs but not the normal cell types. These results also suggested that the binding efficiency was positively correlated to the abundance of cell surface CD44. Moreover cytotoxicity assay revealed that the carrier HA-ADH-PLA was toxic free and suitable for hepatofibrosis treatment. The IC50 of curcumin reduced 30-fold (from 80.4 μM reduce to 2.6 μM) by using the nanoformulation on aHSC, whereas no toxicity observed on other cell types (hepatic cells, clone 9, qHSC). These results indicated the excellence of curcumin-encapsulated HA-ADH-PLA nanoformulation. In summary, phenolic compounds can exert their effects on the eradication of aHSC, especially by nanoformulation.

並列關鍵字

Quercetin ； Gallic acid ； Hyaluronic acid ； Polylactic acid ； Nanoparticle ； CD44 ； Liver fibrosis

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