腫瘤胚基因 (oncofetal gene) 係為表現於胚胎時期的基因，隨著發育為正常成體會降低表現或是不表現，而後當腫瘤形成時，其腫瘤胚基因又再次大量表現。一般較為人熟知的腫瘤胚基因有A型胎兒蛋白 (alpha-fetoprotein, AFP) 及聚醣蛋白3 (Glypican-3, GPC3)。在臨床對肝癌的常規診斷中，往往以偵測血清中AFP蛋白表現來做篩檢。而發展中的腫瘤會持續釋放惡性腫瘤細胞進入血液中，少數游離在血液循環的腫瘤細胞往往能夠躲避體內免疫監控 (immune surveillance) 機制的作用進而有可能由周邊血中被偵測到。在先前有研究顯示從癌症患者周邊血中，可以發現到游離於血液中腫瘤細胞的存在進而有可能成為篩檢上的依據之一；另外在肝癌患者周邊血及骨髓中也可以偵測到AFP mRNA。我們利用生物資訊結合實驗室建立的篩選方法也找到了已知為腫瘤胚基因的GPC3及具類似腫瘤胚基因特徵的 Lin28B (lin-28 homolog B) 與 Zic2 (Zinc finger protein of the cerebellum 2)。在本研究中，我們目的想要建立一套方法，透過採集周邊血，偵測血液中肝癌腫瘤細胞所表現的腫瘤胚基因來達到診斷的目的。首先在我們模擬的血液中肝腫瘤細胞，我們利用即時定量聚合酶反應 (Real-time PCR) 測定腫瘤胚基因表現量來評估出偵測的極限；而後我們收集31位非肝病志願者、7位肝硬化、20位HBV與HCV肝炎、以及34位肝癌患者的周邊血，評估腫瘤胚基因在此三種患者中表現量的差異。我們以已知腫瘤胚基因AFP初步完成所建立的流程再依序完成 GPC3、Zic2、Lin28B在模擬與臨床樣本中的表現。結果發現在模擬之下，10^7白血球細胞數的全血混入人類肝癌細胞株HepG2，其偵測到腫瘤胚基因的極限約在一個細胞數。在臨床檢體的表現方面，我們從非腫瘤檢體所測定的最高表現設定門檻值，結果發現AFP唯能表現在一位肝癌患者，另外Zic2及Lin28B則可以各自在3位及5位肝癌患者中被偵測到。AFP偵測表現並未如預期，但結合另外兩種基因共同偵測的方式相較於偵測單一基因仍具前瞻性。接著未來我們希望能夠利用multiplex PCR的方式在單一反應中增加檢體模板量來有效提升偵測上的敏感性，讓我們所發現的基因與建立的方法可以有效能地投入臨床應用。

Oncofetal genes are generally defined as genes in embryonic cells, down-regulated in normal adult cells, but re-expressed in tumor cells, such as AFP (alpha-fetoprotein) and GPC3 (Glypican-3). In the routine hepatoma diagnosis, the determination of serum AFP has been used. Tumors launched malignant cells into the circulation continuously. A small number of circulating tumor cells escaped from the immune surveillance. Evidences showed that the presence of circulating tumor cells in the peripheral blood of cancer patients can be determined. For example, AFP mRNA was detectable in the peripheral blood and bone marrow of hepatoma patients. Recent studies showed that the well-known GPC3 was a marker of hepatic progenitor cells and of early liver lesions. In addition, we found the oncofetal-like Lin28B (lin-28 homolog B) and Zic2 (Zinc finger protein of the cerebellum 2) by screening experiments combined with bioinformatics. In this study, we build a methodology to detect the oncofetal genes in a small amount of circulating HCC tumor cells. At first, we pooled 10^0-10^5 HepG2 cells into the 10^7 WBC-counts normal peripheral blood respectively to determine the limitation then collected the peripheral blood derived from 31 non liver diseased, 7 cirrhosis, 20 hepatitis, and 34 hepatoma patients. The presence of AFP gene was determined by real-time quantitative PCR. Following the strategy of AFP detections, GPC3, Zic2 and Lin28B will be determined in the spiked circulating tumor cells and in the clinical samples. Up to now, the data showed that these oncofetal genes can be effectively detected in one HepG2 cell pooled with blood cells at least. In clinical samples, AFP was detected in only 1 of 34 hepatoma; the Zic2 was determined in 3 of 34 hepatoma; while the Lin28B was detected in 5 of 34 hepatoma cases. Though the detection rate of well-known AFP was low, the combined detection of AFP, Zic2 and Lin28B in circulating HCC cells is notably more promising than each gene alone. However, the detection of GPC3 had no significant differences between tumor and non-tumor cases. We can increase the detect limitation effectively in the peripheral blood by high-throughput real-time quantitative PCR, our oncofetal-like genes may be powerful and convenient markers in potential clinical applications.

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