競爭式定量聚合?連鎖反應法之建立與應用：台灣地區非小細胞肺癌檢體中，neu 基因表現量之研究

Abstract

目前已有多種分子生物技術用來檢測癌症檢體中致癌基因與抑癌基因的變化情形，並由大量的資料中得到一些有趣的關聯。其中包括neu基因的倍增／過量表現（DNA amplification/over expression） 與癌症轉移（metastasis）具有明顯的關聯性，但也有對此研究結果持否定的看法。這是因為所使用的技術無法精確的定量所致，需要以更精確的方法來釐清這個問題。 競爭式定量聚合?連鎖反應法是一種測量RNA中特定基因表現量的方法。放入一已知量、與目標基因相似的內基準RNA與材料total RNA一起參與反應，競爭所有的資源，然後由反應後兩者cDNA量的比值來計算total RNA中目標基因RNA的量。與以往的方法相較，此法具有total RNA使用量少，精確等優點，使其更適合於檢體上的研究。 我參考自Piatak等人的文獻，在實驗室中建立競爭式定量聚合?連鎖反應法，並嘗試改良，以便更減少材料的使用量、並提高其準確性。所做的改良包括，將目標基因與控制組（GB-like）的內基準RNA與材料total RNA的逆轉錄反應合併進行，以及以85℃熱處理逆轉錄產物等等。經以細胞株total RNA為材料，反覆的測試，證明此法具有良好的穩定性，誤差均於20％以下。與條狀點墨雜交法相較，此法亦有較好的靈敏度。我並以此法檢測台灣地區非小細胞肺癌檢體中neu基因的表現量。在24個檢體中有4個檢體具有7?4倍不等的過量表現，惟與臨床上癌症的轉移並無明顯的關聯性。另外，與先前實驗室中檢測p53基因突變的資料相較，發現neu基因的過量表現與p53基因的突變並不會同時發生，可能是兩者均為癌化現象的重要因數，其一突變就會使致癌機率大增所致。由於收集的檢體量少，所得的結果僅供參考，需要往後的實驗證明。

Quantitative competitive polymerase chain reaction (QC-PCR) is an advanced method to quantitate the amount of specific RNA in cell. The major principle is that the known copy number of a synthetic truncated internal standard is introduced with the sample into the reverse transcription reaction mixture and PCR reaction mixture. Because of it's accuracy and small amount of RNA needed, QC-PCR is not only suitable for RNA quantitation from culture cells as well as from clinical specimens. According to previous studies of human lung and breast cancers, several laboratories have reported a significant association between neu gene DNA amplification/over-expression and tumor metastasis, However, this point is still debatable. The main issue is that method used for RNA was less accuracy such as immunohistochemistry (IHC) or DNA/RNA blot hybridization. In order to clearity this puzzle, a more sensitive and accurate technique is desired necessary. Therefore, a modified QC-PCR was set-up in our laboratory according to the protocol from Piatak. After modification, the stability of this method was increased dramatically. The sensitivity was examined by comparison with slot blot hybridization. Almost one order difference between QC-PCR and slot blot hybridization were observed. This method was then applied to analyze the neu status of non-small-cell lung cancers in Taiwan. Four of 24 cases examed have harbored the neu over-expression from four to seven times. However, due to the small sample size, the association between neu over-expression and tumor metastasis was not significant. The other interesting finding was that the p53 mutation and neu over-expression in tumor specimens examined were exclusively existed. The report here was just a preliminary data, further investigation will be necessary to elucidate the association between neu and metastasis as well as the biological role of p53 mutation/neu over-expression in neoplastic malignance.