Lu Chao, Wu Sheng-hua, Chen Ji-qing, Zhao Fei, Chi Xia, Pan Xiao-qin, Fei Li, Guo Mei, Huang Song-ming, Guo Xi-rong, Chen Rong-hua
Department of Pediatrics, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China.
Zhonghua Er Ke Za Zhi. 2005 Jul;43(7):503-6.
Second mitochondria-derived activator of caspase (Smac) is a recently identified, novel pro-apoptotic molecule, which is released from mitochondria into the cytosol during apoptosis. Smac promotes activation of caspases by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The objective of the study was to examine the pro-apoptotic effect of human Smac gene on Burkitt's lymphoma Raji cells.
The full length cDNA of human Smac gene was amplified by reverse transcription-PCR from total RNA of HEK-293 cells. The PCR product was ligated with linearized vector pGEM-T-easy supplied in the TA cloning kit and sequenced. The correct cDNA of full length Smac was subcloned into eukaryocytic expression vector pcDNA3.1/myc-his and transfected into human Burkitt's lymphoma cell line Raji by lipofectamine-mediated transfection. The expression of full length Smac was determined by Western blot. Morphological observation was done with the laser scanning confocal microscope by double staining the Raji cells with Hoechest 33,258 and propidium iodide. Flow cytometry was used to evaluate apoptosis. Relative caspase-3 activity was determined by colorimetric assay.
Recombinant eukaryocytic expression vector pcDNA3.1/Smac, which contained full length Smac, was successfully constructed. After pcDNA 3.1/Smac was transfected into human Burkitt's lymphoma Raji cell line for 24 hours, Raji cells showed apparent apoptosis with a percentage of (43.7 +/- 2.5)%, which was higher than that of non-transfected group and free vector-transfected group (P < 0.05). Compared with non-transfected group (0.136 +/- 0.036) and free vector-transfected group (0.138 +/- 0.026), the relative caspase-3 activity of Raji cells transfected by pcDNA3.1/Smac (0.936 +/- 0.041) was significantly enhanced (P < 0.05).
Transfection and expression of human Smac gene could significantly induce apoptosis of human Burkitt's lymphoma Raji cells. The mechanism is associated with the increase of caspase-3 activity.
第二线粒体衍生的半胱天冬酶激活剂（Smac）是一种最近发现的新型促凋亡分子，在细胞凋亡过程中从线粒体释放到细胞质中。Smac通过中和凋亡抑制蛋白（IAPs）家族成员，如X连锁凋亡抑制蛋白（XIAP），促进半胱天冬酶的激活。本研究的目的是检测人Smac基因对伯基特淋巴瘤Raji细胞的促凋亡作用。
通过逆转录-PCR从HEK-293细胞的总RNA中扩增人Smac基因的全长cDNA。将PCR产物与TA克隆试剂盒中提供的线性化载体pGEM-T-easy连接并测序。将正确的全长Smac cDNA亚克隆到真核表达载体pcDNA3.1/myc-his中，并通过脂质体介导的转染转染到人伯基特淋巴瘤细胞系Raji中。通过蛋白质免疫印迹法测定全长Smac的表达。用Hoechest 33258和碘化丙啶对Raji细胞进行双重染色，通过激光扫描共聚焦显微镜进行形态学观察。采用流式细胞术评估细胞凋亡。通过比色法测定相对半胱天冬酶-3活性。
成功构建了包含全长Smac的重组真核表达载体pcDNA3.1/Smac。将pcDNA 3.1/Smac转染到人伯基特淋巴瘤Raji细胞系24小时后，Raji细胞出现明显凋亡，凋亡率为（43.7±2.5）%，高于未转染组和空载体转染组（P<0.05）。与未转染组（0.136±0.036）和空载体转染组（0.138±0.026）相比，pcDNA3.1/Smac转染的Raji细胞的相对半胱天冬酶-3活性（0.936±0.041）显著增强（P<0.05）。
人Smac基因的转染和表达可显著诱导人伯基特淋巴瘤Raji细胞凋亡。其机制与半胱天冬酶-3活性增加有关。
