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【目的】探讨自杀基因 pCEAcd tk/前体药物体系旁观者效应的细胞死亡机制。【方法】提取质粒pCEAcd tk ,转染到肺癌细胞株SPC A 1中 ,与未转染的细胞按 2∶8的比例混合后加到 96孔培养板中 (每孔 3× 10 3 ) ,加前体药物 5 FC(5 μmol/L)和GCV(10 μmol/L)孵育 5d后 ,MTT法测定细胞存活率而估计旁观者效应。取处理后的细胞 ,提取DNA ,琼脂糖凝胶电泳 ;Hoechst染色后荧光显微镜观察及碘化丙啶 (PI)染色 ,流式细胞仪分析细胞周期。【结果】①SPC A 1细胞株在转染了pCEAcd tk体系后加前体药物存在明显的旁观者效应 ;②处理细胞的基因组DNA琼脂糖凝胶电泳呈涂布状 ;③处理细胞用荧光显微镜观察到形状变大 ,无凋亡小体形成 ;④流式细胞仪分析到细胞周期发生改变。【结论】SPC A 1细胞在转染了pCEAcd tk后存在旁观者效应 ,这种旁观者效应引起的细胞死亡是一种坏死。
【Objective】 To investigate the cell death mechanism of bystander effect of suicide gene pCEAcd tk / prodrug system. 【Method】 The plasmid pCEAcd tk was extracted and transfected into the lung cancer cell line SPC A1. The cells were mixed with untransfected cells at a ratio of 2: 8 and then added to a 96-well culture plate (3 × 10 3 per well) After 5 days FCM (5 μmol / L) and GCV (10 μmol / L) incubation, cell viability was measured by MTT assay to estimate bystander effect. The treated cells were extracted, DNA was extracted and subjected to agarose gel electrophoresis. Fluorescent microscopy and propidium iodide (PI) staining were performed after Hoechst staining. Cell cycle was analyzed by flow cytometry. 【Results】 ① SPC A 1 cells transfected with pCEAcd tk system after the addition of prodrugs there is a significant bystander effect; ② treated cells with DNA gel electrophoresis was coated; ③ treated cells were observed by fluorescence microscopy To shape larger, no apoptotic body formation; ④ flow cytometry analysis of the cell cycle changes. 【Conclusion】 Bystander effect exists in SPC A 1 cells after transfection with pCEAcd tk, and cell death caused by bystander effect is a kind of necrosis.