應用核糖核酸干擾作用探討細胞內 Haptoglobin 之抗氧化能力

Abstract

Haptoglobin 為人體內的一種分泌性醣蛋白，主要由肝臟合成，為一個抗氧化分子。因為 Hp 和血紅素有強大的結合能力。當血管內部溶血時，能清除血液中游離的血紅素。然而，Hp 在細胞內所扮演的角色仍然無法釐清。本研究主要方針為探討是否 Hp 在肝細胞（HepG2 cells）內的之表現量下降以後，可以改變細胞對於氧化壓力之耐受性。我們的策略是使用小片段干擾核酸（siRNA）這個技術來降解 Hp 在細胞內的合成。經過設計及比對後，我們找到兩段 siRNA 之標的序列，分別位於 Hp α1β 序列上的第 729-747、第987-1005號核甘酸上，再把綠色螢光基因（GFP）接在 pSUPER 載體上作為報告基因。偵測 siRNA 在細胞內表現之情形。藉由流式細胞儀的實驗顯示 siRNA 的確能進入細胞，我也利用免疫螢光染色法證實 此段 siRNA 的確能顯著地減少HepG2 細胞中 Hp 之表達。再者也發現被轉染 siRNA 後之細胞與未轉染及轉染空載體後的細胞相比，其抗氧化能力下降了約 50%。由上可知，我們證明了 Hp 在對抗細胞遭受氧化性傷害之過程裡，扮演非常重要的角色。

Haptoglobin, an antioxidant molecule, is a secretive glycoprotein primarily synthesized in the liver. Because of its great binding affinity with blood hemoglobin, Hp plays an important role in removing free hemoglobin of intravascular hemolysis. The cellular function of Hp, however, is unknown and remains elusive. The present goal is to test the hypothesis whether the low level of Hp in hepatocytes（HepG2 cells） may attenuate the tolerance against oxidative stress. The strategy was to use small interfering RNA（siRNA）to knockdown the Hp synthesis. After a blast search, two siRNAs corresponding to nucleotide sequence 729-747 and 987-1005 were selected as a target for the construction of a plasmid containing green fluorescence protein（GFP）gene as a reporter on a pSUPER vector. Flowcytometric analysis revealed that trasfection of siRNAs was achieved. Immunocytochemical study demonstrated the expression of Hp being significantly reduced in HepG2 cells. The antioxidative stress of these cells were markedly attenuated by approximately 50% as compared to cells either nontransfected or transfected with vector only. Our data suggest that Hp plays a crucial role against cellular oxidative stress.