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目的克隆人促凋亡基因(secondmitochondriaderivedactivatorofcaspases,Smac),研究Smac基因转染对Burkitt′s淋巴瘤Raji细胞的促凋亡作用。方法从人胚肾293细胞中扩增人Smac基因全长的cDNA;构建含Smac基因全长cDNA的真核表达载体pcDNA3.1/Smac,并转染人Burkitt′s淋巴瘤Raji细胞;用Westernblot测定外源基因的表达;Hoechest33258和碘化丙锭双荧光染色,结合激光共聚焦显微镜观察细胞凋亡形态;流式细胞仪分析细胞凋亡百分率;比色法测定caspase3酶活性。结果成功克隆人Smac基因全长cDNA,并构建真核表达载体pcDNA3.1/Smac;转染人Smac基因全长cDNA的真核表达载体24h,可显著诱导Burkitt′s淋巴瘤Raji细胞的凋亡,凋亡百分率达(43.7±2.5)%;转染人Smac基因全长cDNA的真核表达载体24h,可显著诱导Raji细胞中caspase3酶活性上调,吸光度为0.936±0.041,与正常对照组(0.138±0.026)及空载体转染对照组(0.136±0.036)相比,P<0.05。结论转染并表达外源性Smac基因,可显著诱导人Burkitt′s淋巴瘤Raji细胞凋亡。其机制可能与caspase3酶活性上调有关。
Objective To clone the pro-apoptotic gene (Smac) and to investigate the pro-apoptotic effect of Smac gene on Burkitt’s lymphoma Raji cells. Methods The full-length cDNA of human Smac gene was amplified from human embryonic kidney 293 cells. The eukaryotic expression vector pcDNA3.1 / Smac containing Smac full-length cDNA was constructed and transfected into Burkitt’s lymphoma Raji cells. Western Blot The expression of exogenous gene was measured. Double staining with Hoechest 33258 and propidium iodide staining was used to observe the morphology of apoptotic cells by laser confocal microscopy. The percentage of apoptotic cells was analyzed by flow cytometry. The activity of caspase3 was assayed by colorimetric assay. Results The full-length cDNA of human Smac gene was successfully cloned and the eukaryotic expression vector pcDNA3.1 / Smac was constructed. The transfection of the full-length cDNA of Smac gene in eukaryotic expression vector for 24 hours significantly induced the apoptosis of Burkitt’s lymphoma Raji cells (43.7 ± 2.5)%. The expression of caspase3 in Raji cells was significantly up-regulated after transfected with the full-length cDNA of human Smac gene for 24 hours. The absorbance was 0.936 ± 0.041, which was significantly higher than that in the normal control group (0.138 ± 0.026) and empty vector control group (0.136 ± 0.036), P <0.05. Conclusion Transfection and expression of exogenous Smac gene can significantly induce apoptosis of Raji cells in human Burkitt ’s lymphoma. The mechanism may be related to the up-regulation of caspase3 activity.