促癌变剂对淋巴细胞核仁rRNA基因转录的活化作用——rDNA原位杂交荧光显示法

ACTIVATION OF NUCLEOLAR rRNA GENE TRANSCRIPTION IN LYMPHOCYTES BY TUMOR PROMOTERS STUDIES WITH INSITU HYBRIDIZATION DETECTED BY FLUORESCENT METHOD

用原位杂交荧光显示法观察了人淋巴细胞在促癌变剂黄芫花提取物(WCE)和12-0-十四烷巳豆醇-13乙酸酯(TPA)处理后,间期核仁rDNA的定位与数量改变,并与丝裂原植物血细胞凝集素(PHA)的效应作了比较,同时用银染色法观察了核仁。对照组淋巴细胞核仁小,原位杂交的rDNA为少数明亮荧光斑和分散的荧光点。经促癌变剂WCE和TPA处理后,银染色的核仁增大,银染颗粒增多,表明rDNA转录活化。原位杂交证明rDNA信号数目明显扩增,许多荧光小点断续相连形成网织状结构,与PHA刺激核仁转录活化的表现一致。对核仁内所含银染颗粒和rDNA荧光斑点数均值的统计学分析表明,WCE、TPA和PHA各加药组均明显多于对照组。3个加药组之间无明显差别。提示两种促癌变剂皆具有刺激核仁rDNA扩增和转录活化的效应。

Effects of tumor promoters on nucleolar rRNA gene transcription in human lymphocytes were studied by using rDNA non-autoradiographic in situ hybridization (ISH) fluorescent detection and silver (Ag) staining methods. In untreated control lymphocytes, ISH revealed a few rDNA fluorescent agglomerations of spots. Ag-staining showed only one small nucleolus. After exposure to tumor promoting agents wikstroemia chamaedaphne extract (WCE, 40mg/L) and 12-0-tetradecanoyl-13-phorbol acetate (TPA, 60μg/L) respectively for 72 hours, ISH showed that rDNA fluorescent spots and dots increased remarkably. These rDNA spots and dots connected to form large reticulate structures. When stained with silver method, more than one nucleoli were observed. These nucleoli were enlarged in size and contained increased amount of Ag-particles. The above changes suggested that activation of nucleolar rRNA gene transcription and rRNA gene amplification be in duced. The changes induced by WCE and TPA resemble changes after Phytohemagglutinin (PHA, 80mg/L, 72h) stimulation in lymphocytes. Statistic analysis of the countings of Ag particles and rDNA spots/dots per cell proved significant difference between control and drug (WCE, TPA, or PHA) treated groups respectively.