從石蠟包埋肺癌組織提取RNA 檢測PML 基因轉錄的研究 趙志龍 宗志紅 李建新 劉宏旭 趙惠儒 【摘要】 背景與目的 甲醛固定石蠟包埋組織（FPE）包含大量臨床病理信息，同時還含有大量的基因和蛋 白物質可用來研究臨床意義。本研究的目的是探討從FPE中提取RNA的可能性，檢測急性早幼粒細胞白血病基因 （PML）在肺癌FPE中的轉錄表達。方法 選取前期經免疫組織化學染色證實無PML蛋白表達的40例肺癌FPE，存檔 時間在60-85個月，5例新鮮冰凍肺腺癌組織作為對照；利用Trizol一步法提取RNA，經OD 值檢測驗證RNA質量；RTPCR 檢測PML的基因轉錄表達。結果 自肺癌FPE和新鮮冰凍組織提取的RNA的OD 比值在1.7-2.1。RT-PCR顯示，40 例FPE以及5例新鮮冰凍組織中，PML（295 bp）以及β-actin（318 bp）mRNA均有表達。結論 利用Trizol一步法自 FPE提取RNA進行RT-PCR，可有效擴增出300 bp左右的產物；肺癌組織中PML蛋白存在轉錄后缺失。 【關鍵詞】 RNA提取 石蠟包埋組織 人PML蛋白 RT-PCR 病理學 【中圖分類號】 R734.2 DOI:10.3779/j.issn.1009-3419.2008.04.010PML expression in lung carcinomas: analysis by RT-PCRin formalin-fixed and paraffin-embedded tissueZHAO Zhilong*#, ZONG Zhihong** , LI Jianxin# , LIU Hongxu*, ZHAO Huiru**Department of Thoracic Surgery, First Hospital, China Medical University,Shenyang, Liaoning 110001, China;**Department of Biochemistry, College of Basic Medical Science, China Medical University, Shenyang, Liaoning 110001,China; #Department of Thoracic Surgery, Zhongshan Hospital, Dalian University, Dalian, Liaoning 116001, China Corresponding author: ZHAO Huiru, E-mail: xuhua1998@163.com 【Abstract】 Background and objective Formalin-fixed and paraffin-embedded tissue (FPE) with long termfollow-up and clinicopathological data are extensively available and easily accessible. They represent an extensive sourceof genetic materials and proteins to be investigated for clinical usage. We conducted the study to explore the possibilityof extracting RNA from FPE of lung cancer, and checking up the transcription levels of the promyelocytic leukemia gene(PML) by RT-PCR. Methods five fresh frozen lung adenocarcinoma tissues and 40 FPEs of lung cancer were chosen; FPEs had been conserved for 60-85 months. All samples had been identified lacking expression of PML protein byimmunohistochemical staining. The total RNA was extracted by using Trizol one-step method, and verified by testing ODvalue. RT-PCR was used to detect the transcription level of PML gene. Results The RNAs from both tissues had the ODvalue ranging from 1.7 to 2.1. RT-PCR results displayed that mRNA of PML (295 bps) and β-actin (318 bps) was notabsent in all cancer tissues. Conclusion Trizol one-step method is easily accessible, and reliable for extracting RNAin fragments around 300 bases from FPEs. Loss of PML protein in lung cancer is not caused by downward adjustment ofmRNA. 【Key words】 RNA extraction Paraffin-embedded tissue Human PML protein RT-PCR Pathology 越來越多的研究證明[1-4]：自甲醛固定-石蠟包埋組織（Formalin-fixed and paraffin- embedded tissue，FPE）可以提取到RNA，并能用逆轉錄聚合酶鏈反應（RT-PCR）檢測基因轉錄表達，甚至可以進行基因表達定量分析。這個方法學的創立及完善對于發現和發展新的診斷方法和治療藥物具有非常大的實際價值。現已證實基因表達譜與疾病預后相關，研究人員正在尋找可以預測疾病預后或治療反應的新基因亞群[5]。而腫瘤分子分期也不斷被得到認可和推廣。目前，FPE是最廣泛的、包含臨床預后資料的實體組織樣本庫，如何充分利用并解讀其所包含的寶貴信息尤需重視。

本研究探討了Trizol一步法自FPE提取RNA的可能性和可靠性；同時，針對PML蛋白無表達的肺癌組織[6]，檢測其基因轉錄水平的情況。

1 材料 1.1 組織來源 選取1998年1月-2000年6月外科手術切除的肺癌FPE標本40例[6]，其中小細胞肺癌10例，非小細胞肺癌30例（腺癌15例，鱗癌15例），肺癌組織手術切除后常規經10%中性甲醛固定、石蠟包埋存檔。5例手術切除后新鮮冰凍肺腺癌組織作為對照。所有組織前期均經免疫組織化 學染色證實無PML蛋白表達[6]，見圖1。入選病例均無術前放療或化療。