- 學位論文
腦型肝醣磷酸化酶作為大鼠肝臟前驅細胞新穎標記之鑑定與研究
Identification and characterization of brain isoform glycogen phosphorylase as a novel marker of rat hepatic progenitor cells
摘要
肝臟雖然具備再生的能力，然而當切除或受損超過三分之二以上時，肝臟細胞的再生能力也會受到抑制，一般認為在此狀況下，肝臟前軀細胞（hepatic progenitor cell）或肝臟幹細胞（liver stem cell）會被激活，進而增殖並分化為成熟的肝臟細胞，取代缺損部分以便修復肝臟功能。多數研究學者認為，低分化的“卵圓細胞”（oval cell）是具有雙效力（bipotency）分化能力之肝臟前軀細胞，可以分化成膽管上皮細胞（biliary epithelial cell）和肝細胞（hepatocyte）。到現在為止，至少有4種類型的肝臟前軀細胞被認是候選肝臟幹細胞，包括卵圓細胞，星狀肝細胞（也稱為Ito細胞） ，肝上皮細胞（liver epithelial cell），以及間葉幹細胞（mesenchymal stem cell）。多數研究認為肝上皮細胞保留一些類似幹細胞的特性，且被證明能夠修復嚴重損傷或切除之肝臟部分。其中研究最多的是從成年雄性Fischer-344大鼠肝臟分離出來的WB-F344細胞，因為它們具有肝臟前軀細胞小而多角形的形態特徵，同時表達肝細胞系標記物甲型胎兒蛋白、白蛋白和膽管細胞系標記物細胞角質蛋白，而且它們不僅能分化成肝細胞和膽管上皮細胞，在給予特定生長因子時，他們也能接收訊息分化成其他細胞。此外從雄性Fischer-344大鼠肝臟非實質部分分離出的Lig-8細胞也與WB-F344細胞型態相似且具有分化成肝細胞和膽管上皮細胞之潛能。然而人類肝臟幹細胞卻一直沒能被成功的分離出來，主要是缺乏獨特可利用的標記來分離。本研究證明Lig-8細胞株被證明能夠在丁酸鈉（sodium butyrate）誘導下分化成肝細胞和膽管細胞，並且表達這些終端成熟細胞的標記：白蛋白和細胞角質蛋白19 。我們利用Lig-8細胞作為免疫原刺激BALB/c小鼠，生產並純化對Lig-8細胞具有專一特異性的單株抗體，命名為Ligab。藉由免疫螢光染色法與流式細胞儀的分析，我們發現Ligab不僅能專一的辨認肝臟前軀細胞Lig-8，也一樣可以專一辨認WB-F344細胞，我們將Ligab辨認的抗原稱為Ligab Ag。實驗結果顯示，Ligab無法在正十二烷硫酸鈉（sodium dodecyl sulfate, SDS）濃度超過2%以上的環境下辨認Ligab Ag，這是一般標準的正十二烷硫酸鈉-聚丙烯醯胺膠電泳（sodium dodecyl sulfate-polyacrylamide gel, SDS-PAGE）所含SDS的濃度。因此我們推論Ligab所辨認的抗原分子可能是分子結構，而不是一個胜肽序列。因此本研究進一步利用免疫沈澱法來抓取Lig-8細胞膜蛋白質萃取液中的Ligab Ag，藉由標準的SDS-PAGE電泳和銀染分離出有興趣的候選蛋白胜肽片段，以液相層析串聯式質譜（Liquid Chromatography Combined Tandem Mass Spectrometry, LC-MS/MS）分析胜肽片段，從資料庫中比對出肝醣磷酸化酶（glycogen phophorylase）可能是Ligab認得的抗原之一。肝醣磷酸化酶是一種肝糖分解代謝中的關鍵酶，催化肝糖的磷酸分解反應，此種酶有三種異構酶，分別是腦型、肝臟型與肌肉型，其中腦型肝糖磷酸化酶（brain isoform glycogen phosphorylase, GPBB）最早出現於胚胎發育中， 因此胚胎型肝醣磷酸化酶（fetal isoform of glycogen phophorylase）被認為是腦型肝糖磷酸化酶。經由逆轉錄聚合酶鏈式反應與西方點墨法證實，肝臟前軀細胞Lig-8和WB-F344都大量表達腦型肝醣磷酸化酶，卻不表達肝臟型或肌肉型的肝醣磷酸化酶。有趣的是當我們用丁酸鈉來誘導Lig-8細胞分化時，GPBB被表達的量驟減，取而代之的是肝臟型的肝醣磷酸化酶。進而，我們設計以小髮夾型核醣核酸（short hairpin RNA; shRNA）來沉默WB-F344細胞中的GPBB基因表達，再以丁酸鈉來誘導細胞分化，結果顯示GPBB基因沉默會延遲WB-F344細胞的分化，顯示GPBB在此類前驅細胞的分化上扮演某種角色。而且GPBB的基因沉默也影響細胞在缺少葡萄糖的培養基中的存活率。也就是說被干擾GPBB蛋白表達的細胞，其死亡率有意義的高於GPBB正常表達的細胞，此種現象，在相對低濃度（1 g/mL）葡萄糖細胞培養基或移除葡萄糖與丙酮酸鈉（sodium pyruvate）的細胞培養基中，更明顯。 我們認為GPBB不僅可以作為肝臟前軀細胞的新穎性標記，而且它在肝臟前軀細胞的分化上可能扮演某種角色。在缺少能量的環境中，GPBB可以幫助前軀細胞的存活。
並列摘要
The liver is constantly renewing itself under normal conditions. With adult stem cells having been discovered in many of the body's organs, one might assume that the tremendous regenerative capacity of the liver is contributed by liver stem/progenitor cells. Until now, at least four types of liver cells are considered as candidate of liver stem cells, including oval cells, hepatic stellate cells (also known as Ito cells), liver epithelial cells (liver epithelial cell), mesenchymal stem cells. However, it hasn’t been so easy to isolate in mammals due to limited liver progenitor cell markers. An adult rat liver progenitor cell line Lig-8 has been established, which is characterized like liver epithelial cell. In the induction of sodium butyrate, these cells were shown able to differentiate into both hepatocytes and bile duct cells expressing albumin and cytokeratin-19, the markers of respective cell types. We previously generated a monoclonal antibody using the whole Lig-8 cells as immunogen. The yielded monoclonal antibody, named as Ligab, belongs to IgG subclass G1 and κ-light chain. It specifically reacted to Lig-8 cells but it did not react to a rat hepatoma cell line H4IIE. Furthermore, the expression of Ligab antigen in Lig-8 cells declined under sodium butyrate (SB)-induced differentiation was found. We took advantage of Ligab antibody to immunoprecipitate the target antigen, and identify glycogen phosphorylase (GP) in Ligab immunoprecipitates by using liquid chromatography combined with tandem mass spectrometry. Three GP isoforms exist in mammals: the brain isoform (GPBB), liver isoform (GPLL), and muscle isoform (GPMM). Three isoform-specific polyclonal antibodies and antiserum were used to examine the existence of these GP isoforms in the liver progenitor cells. Immunoblotting results showed that GPBB was expressed in two liver progenitor cell lines Lig-8 and WB-F344. The level of GPBB expression decreased in the WB-F344 cells under sodium butyrate (SB)-induced cell differentiation, which was consistent with roles of GPBB as a liver progenitor cell marker. The short hairpin RNA (shRNA)-mediated GPBB knockdown followed by glucose deprivation test shows that GPBB aids in liver progenitor cell survival under the low glucose conditions. Furthermore, shRNA-mediated GPBB knockdown followed by SB-induced cell differentiation shows that reducing GPBB expression delayed liver progenitor cell differentiation. In conclusion, GPBB is a novel liver progenitor cell marker, which facilitates liver progenitor cell survival under the low glucose conditions and cell differentiation.
參考文獻
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