逆转录病毒介导野生型P^53与反义mdm2融合基因对Mc3细胞凋亡的影响

P^53 gene ;antisense mdm2 gene ;transmission electron microscopy ;cell apoptosis

目的探讨P^53与反义mdm2 cDNA序列真核表达载体诱导人黏液表皮样癌高转移细胞株Mc3细胞凋亡的影响。方法将P^53与反义mdm2 cDNA序列真核表达载体通过脂质体转染Mc3细胞，采用形态学观察凋亡细胞；琼脂糖凝胶电泳检测“梯状条带”；末端转移酶标记法（TUNEL）检测凋亡指数；透射电镜检测凋亡小体。结果转染48h后形态学观察到细胞体积缩小；核浓染碎裂成大小不等的碎片；核染色质成新月形，琼脂糖凝胶电泳可见180～200bp典型的凋亡带，TUNEL检测凋亡指数为25．22±1．01（转染后），与对照组2．01±1．10（转染前）比较差异有统计学意义（P〈0．05），转染空载体（2．02±1．11）与对照组比较差异无统计学意义（P〉0．05），透射电镜可见染色质浓集分布不均，形成由核膜包裹断裂的核碎片的凋亡小体。结论P^53与反义mdm2基因融合体真核表达载体能诱导和促使体外培养的人黏液表皮样癌高转移细胞株Mc3细胞发生凋亡。

Objective To discuss the eukaryotic expression vector containing P^53 and mdm2 antisense cDNA sequence induce apoptosis in human mucoepidermoid carcinoma high metastatic cell strain Mc3. Methods To put the eukaryotic expression vector containing P^53 and mdm2 antisense cDNA sequence into Mc3 cell by utilizing liposome transfection. The apoptosis was observed by morphology;The ＂ladder strap＂ was detected by agarose gel electrophoresis ;the apoptosis index was detected by TUNEL and the apoptotic bodies was detected by transmission electron microscope. Results 48 h after transfection,the morphology showed that the cell volume was diminution; the nuclear was strongly stained and cracked into small pieces,the nuclear chromatin was margination as a crescent. Agarose gel electrophoresis showed apoptosis band at 180-200 bp. TUNEL detection showed that the difference of the apoptosis index between the group after transfection （25.22±1.01） and the control group before transfection （2.01±1.10） was significant（P〈0. 05）. It had no statistical dif- ference between the sham group（2.02±1.11） and the control group（P〈0. 05）. Transmission electron microscopy showed dense aggregation of chromatin and the apoptotic bodies. Conclusion The eukaryotic expression vector containing pss and mdm2 antisense cDNA sequence can induce apoptosis in human mucoepidermoid carcinoma high metastatic cell strain Mc3.