肝癌是一種常見的惡性腫瘤普遍發生於全世界。在台灣，肝癌更是高居所有癌症死亡原因中的首位。流行病學的研究中發現，肝癌的發生與慢性B型肝炎病毒 ( HBV hepatitis B virus ) 的感染有極大的相關性。然而，B 型肝炎的感染與誘發人類肝癌的發生過程中，究竟是透過何種機制 ? 這方面相關的研究在目前並不是相當清楚。近年來，有研究文獻認為是由病毒所產生的一些蛋白質，如:
HBxAg 及 HBsAg 藉由蛋白質與蛋白質間的交互作用，而促進肝癌的形成。更進一步在動物實驗的研究發現 HBsAg 的確可以在轉殖基因鼠中，導致肝癌的發生。
為了進一步釐清細胞中有哪些蛋白質可以與 HBsAg，藉由蛋白質間的交互作用促使肝癌形成。因此我們實驗室採用Yeast two hybrid 的技術，在人類肝細胞的cDNA 基因庫中，大規模的篩選與 HBsAg 有交互作用的蛋白質，在篩選了約 1x 107個轉型的酵母菌菌落中，僅有267個菌落可以在有缺陷的培養基中生長，並且在 X-gal 上呈現藍色。而在這 267 個菌落經過核酸定序，且利用 NCBI (National Center for Biotechnology Information) 的核酸-蛋白質資料庫系統分析的結果發現，只有19個基因可以與 GAL4-AD 具有相同的轉譯起始區， 比對到有意義基因。在這些基因中有一部分是與代謝相關的蛋白質。除此之外，在 Yeast two hybrid 的系統，我們利用了共同轉型及酵母菌融合的技術，更進一步的測試蛋白質交互作用的情形。發現僅有Apolipoprotein H (ApoH) 及 acid α-glucosidase 能與 HBsAg 有交互作用。而在有缺陷的培養基上生長，且使得 X-gal 呈現藍色。 然而早在1994年即有文獻報告證明，血清中的 ApoH 會與 HBsAg 有交互作用。
因此，我們實驗室欲加以探討 acid α-glucosidase 與 HBsAg 的蛋白質交互作用情形，及其功能性的研究。首先我們先利用 RT-PCR（Reverse transcriptase- polymerase chain reaction）的技術，由 HepG2 cells 的mRNA中經由反轉錄酵素的作用獲得α-glucosidase 的 cDNA 片段其全長約為 2859 bp，再將其全長及數個truncated form，分別建構在哺乳細胞表現的載體 pcDNA 3.0。我們已由結果細胞外轉譯/轉錄的 TNT 分析法，以及細胞內免疫沉澱法-ELISA的實驗結果證實， acid α-glucosidase 會藉由其 C 端區域與 HBsAg 產生交互作用。
一般觀念認為當正常細胞受外在刺激，將轉型成癌細胞時會改變其細胞內正常的醣類代謝。 Acid α-glucosidase 在肝細胞中扮演著將肝醣代謝成葡萄糖的重要角色。進一步研究 acid α-glucosidase在生物體內的重要性，我們已經在一些穩定表現 HBsAg 的肝細胞中發現，無論是細胞內的肝醣含量，抑或是α-glucosidase 的活性，在 HBsAg 存在的情況下，都會有明顯的改變。這兩個形成交互作用的蛋白質在細胞中分佈的情形，我們也接著利用免疫螢光顯微鏡觀察。由上述的結果顯示 HBsAg 可以藉由α-glucosidase 的 C 端區域與之結合，而且可能改變肝細胞原本醣類的代謝，而促使肝癌的形成。

Hepatocellular carcinoma (HCC) is one of the most common fatal malignant tumors worldwide and ranks as the first leading course of cancer death in Taiwan. Epidemiological studies have provided strong evidence for the link between chronic Hepatitis B virus (HBV) infection and the induction of HCC. However, many questions about the mechanism of HBV-related hepatocarcinogenesis in human remained unknown. Some studies have been suggested that the viral products of HBxAg and HBsAg play an important role in the development of HCC. Furthermore, there are some evidences shown that HBsAg is highly associated with HCC in transgenic mice.
In an attempt to identify cellular factors that can interact with HBsAg, we performed the yeast two-hybrid system for large-scale screening in human liver cDNA library. Of 1x107 transformed colonies screened, about 267 clones grew in the selective media and were X-gal positive. Among the 267 clones sequenced and alignment to NCBI database, only 19 clones that contained in frame sequence with GAL4-AD protein. Parts of them are associated with metabolic pathway. Further retesting protein interactions in yeast by cotransformation or yeast mating, only two clones were still positive-ApoH and acid α-glucosidase. ApoH has been reported to interact with HBsAg in previous reports.
Changes in carbohydrate metabolism are a hallmark of malignant transformation acid α-glucosidase plays a key role in converting glycogen to glucose. The biological significance of the interaction between HBsAg and acid α-glucosidase was demonstrated by the alteration of glycogen breakdown and α-glucosidase activity in the cells expressing HBsAg.
To characterize the physical interaction between α-glucosidase and HBsAg, we have cloned the full-length cDNA of α-glucosidase from mRNA of human HepG2 cells by RT-PCR. The full-length and various truncated α-glucosidase were cloned into the expression vector pcDNA3.0. The interaction between HBsAg and glucosidase was mediated through C-terminal domain of glucosidase as demonstrated by in vitro Transcription-Translation TNT assay and in vivo IP-ELISA. The cellular localization of HBsAg and�n α-glucosidase were further examined by immunofluorescence microscopy. Our results indicated that HBsAg interacted with C-terminal portion of α-glucosidase, and probably alter the carbohydrate metabolism in hepatocytes.

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