从石蜡包埋肺癌组织提取RNA检测PML基因转录的研

从石蜡包埋肺癌组织提取RNA 检测PML 基因转录的研究 赵志龙 宗志红 李建新 刘宏旭 赵惠儒 【摘要】 背景与目的 甲醛固定石蜡包埋组织（FPE）包含大量临床病理信息，同时还含有大量的基因和蛋 白物质可用来研究临床意义。本研究的目的是探讨从FPE中提取RNA的可能性，检测急性早幼粒细胞白血病基因 （PML）在肺癌FPE中的转录表达。方法 选取前期经免疫组织化学染色证实无PML蛋白表达的40例肺癌FPE，存档 时间在60-85个月，5例新鲜冰冻肺腺癌组织作为对照；利用Trizol一步法提取RNA，经OD 值检测验证RNA质量；RTPCR 检测PML的基因转录表达。结果 自肺癌FPE和新鲜冰冻组织提取的RNA的OD 比值在1.7-2.1。RT-PCR显示，40 例FPE以及5例新鲜冰冻组织中，PML（295 bp）以及β-actin（318 bp）mRNA均有表达。结论 利用Trizol一步法自 FPE提取RNA进行RT-PCR，可有效扩增出300 bp左右的产物；肺癌组织中PML蛋白存在转录后缺失。 【关键词】 RNA提取 石蜡包埋组织 人PML蛋白 RT-PCR 病理学 【中图分类号】 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 越来越多的研究证明[1-4]：自甲醛固定-石蜡包埋组织（Formalin-fixed and paraffin- embedded tissue，FPE）可以提取到RNA，并能用逆转录聚合酶链反应（RT-PCR）检测基因转录表达，甚至可以进行基因表达定量分析。这个方法学的创立及完善对于发现和发展新的诊断方法和治疗药物具有非常大的实际价值。现已证实基因表达谱与疾病预后相关，研究人员正在寻找可以预测疾病预后或治疗反应的新基因亚群[5]。而肿瘤分子分期也不断被得到认可和推广。目前，FPE是最广泛的、包含临床预后资料的实体组织样本库，如何充分利用并解读其所包含的宝贵信息尤需重视。

本研究探讨了Trizol一步法自FPE提取RNA的可能性和可靠性；同时，针对PML蛋白无表达的肺癌组织[6]，检测其基因转录水平的情况。

1 材料 1.1 组织来源 选取1998年1月-2000年6月外科手术切除的肺癌FPE标本40例[6]，其中小细胞肺癌10例，非小细胞肺癌30例（腺癌15例，鳞癌15例），肺癌组织手术切除后常规经10%中性甲醛固定、石蜡包埋存档。5例手术切除后新鲜冰冻肺腺癌组织作为对照。所有组织前期均经免疫组织化 学染色证实无PML蛋白表达[6]，见图1。入选病例均无术前放疗或化疗。