仿生肝臟微流道晶片的建立與抑制膽固醇生成藥物Statin之抗癌效果測試

Abstract

Statin是一種降血脂藥物，在經過肝臟細胞色素CYP450-3A4後所產生的鄰烴基代謝物和β-氧化代謝物，仍具有相當的藥物活性，這些代謝物在整個抗血脂反應過程中佔了70%的效果。而這樣的代謝物除了能降低血脂之外，對於罹癌的臨床病人亦有療效。Statin對癌細胞的抑制機制可能是由於其會抑制內源性的膽固醇生成和阻止蛋白質異戊二烯化(Prenylation)來達到阻止癌細胞增生和轉移的治療效果。然而其機制仍不明確，因此本研究利用自行開發的仿生肝臟微流道晶片來模擬體外的微型抗癌藥物代謝工廠，以期能做為高通量細胞毒性檢測的平台。 我們首先針對Statin以及其活性代謝物對癌細胞的作用影響，在in vitro study下進行探討驗證。實驗結果證實經由肝細胞代謝後所產生的活性代謝物會對人類前列腺癌細胞(PC3)及人類肝癌細胞(HepG2)的活性有明顯抑制。而在高濃度Simvastatin(100µM)的添加條件下，經由肝細胞代謝後所產生的活性代謝物則會使PC3及HepG2的生存率分別下降約20%及15%。 另外一方面，肝細胞於本研究開發的仿生肝臟微流道平台的培養環境下，肝的白蛋白分泌量會比在一般培養皿的環境下提高三倍；尿素分泌量則會提高四倍；由基因表達分析檢測結果亦顯示，CYP1A2基因表現量能提高19倍、且CYP3A23的基因表現量亦提高17倍。由上述生化分析結果可以得知，在此仿生肝臟微流道晶片的培養條件下，可以促進肝細胞的生存活性及機能表現。此平台不但能夠良好的模擬體內肝臟生存情況更使其能具備良好的肝機能。 此外，由實驗結果亦證實Statin經由肝臟酵素轉化成活性代謝物後能有效的毒殺PC3、HepG2兩種癌細胞，並且對於初代肝細胞以及纖維母細胞等正常細胞並沒有顯著的傷害。本研究所開發的仿生肝臟微流道晶片不但更符合真實的體內環境，亦能做為體外藥物代謝和毒物研究平台。將來此平台亦希望能夠結合即時細胞攝影的功能，期許能於體外進行一系列且高通量的生化機能、基因表現、以及代謝作用等之模擬分析。相信這樣的研究對於肝組織工程及器官晶片的領域具有很大的開發潛力。

Statin is a competitive inhibitor of HMG-CoA reductase. And HMG-CoA is the enzyme of the mevalonate pathway required for the biosynthesis of cholesterol. In addition to its capacity of lower cholesterol levels and against the cardiovascular disease, statin also consider as a kind of drug of anti-cancer. Some researchers consider statin may induce apoptosis in various cancer cells, but still don’t know its pathway. For this purpose, we developed a microfluidic device that integrated rat hepatocyte and cancer cell on a chip. The aim of this design is to recapitulate the effects of statin in cancer cell. First, we do the preliminary study in vitro to survey the effect of statin in cancer cell. Results show that hepatic metabolism of statin which released from cells in cell culture medium of statin inhibit PC3 and HepG2 cells. HepG2 cells treated with 100 µM hepatic metabolism statin reduced 15% compared to those cells treated with statin. And PC3 cells treated with 100 µM hepatic metabolism statin reduced 20% compared to those cells treated with statin. On the other hand, we demonstrate that rat primary hepatocyte cultured in microfluidic chip can be maintained successfully for 3 days. Besides, we observed the albumin synthesis increase three times and urea excretion increase four times under flow compared to traditional dish. The activity of CYP1A2 gene expression increase 19 times and CYP3A23 gene expression increase 17 times. In conclusion, we propose a proof of concept for recapitulation of the effects of statin an anti-cancer drug in vitro. And we develop a convincing liver-on-chip devise. This liver-on-a-chip should be further evaluated in other drug related studies.