转染自杀基因pCEAcd-tk的SPC-A-1细胞的死亡机制  

The Mechanism of Cell Death in SPC-A-1 Cell Line Transfected with Suicide Gene pCEAcd-tk

作　　者：郭语彬[1] 伍新尧[2] 骆晓枫 李芳[1] 徐平[1] 何光耀[1] 

机构地区：[1]中山医科大学生化教研室 [2]中山医科大学法医学系,广东广州510089

出　　处：《中山医科大学学报》2000年第2期120-122,共3页Academic Journal of Sun Yat-sen University of Medical Sciences

基　　金：在读博士研究生资助! ( 3 3 10 98)

摘　　要：【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 study the mechanism of cell death of SPC A 1 cell line transfected with pCEAcd tk. Methods Plasmid pCEAcd tk was extracted and transfered into the SPC A 1 cell line, then it was mixed with untransfected cells(2∶8) and seeded onto the 96 well (3×10 3 /well). The prodrugs such as 5 FC (5 μmol/L) and GCV (10 μmol/L) were added and then cultured for 5 days. The survival rate of cells was measured by MTT assay, and the bystander effect was determined. Genomic DNA from the treated cells was extracted and subject to electrophoresis in 8 g/L agorose gel. Treated cells were stained by Hoechst 33342 and observed under the fluorescent microscopy, stained by propidium iodide for analysis with flow cytometry. Results ①The bystander effect of pCEAcd tk/prodrug system was observed after transfecting SPC A 1 cell line with pCEAcd tk; ②The genomic DNA from the treated cells in electrophoresis in 8 g/L agorose gel showed smearing. ③The treated cells were enlarged but no apoptotic body was observed under fluorescent microscopy when they were stained by Hoechst 33342 . ④S stage of treated cells was longer than that of untreated cells, it showed that there were some changes of cellular cycle in SPC A 1 cells transfected with pCEAcd tk. Conclusion There were bystander effect in SPC A 1 cell transfected with pCEAcd tk, and it was necrosis rather than apoptosis.

关 键 词：自杀基因 旁观者效应 pcEAcd-tk SPC-A-1 肿瘤 

分 类 号：R73－36[医药卫生—肿瘤]