【摘要】 目的观察生存素反义脱氧寡核苷酸(antisense oligodeoxynuclecotide,ASODN)诱导人喉癌细胞系Hep2凋亡的作用及对喉癌动物模型抑瘤率的影响,以探讨反义技术选择性封闭目的基因表达进而实现抗肿瘤的作用,为喉癌的基因治疗提供新思路。方法用脂质体LipofectamineTM2000将反义寡核苷酸生存素片段导入人喉癌细胞系Hep2,通过测定细胞增殖的四甲基偶氮唑蓝[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide,TMM]法检测转染后72h内的细胞生长情况;转染48h后逆转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)检测生存素基因表达,Western Blot检测蛋白表达;同时细胞凋亡原位检测(terminal deoxynucleotidemediated nick end labeling,TUNEL)法和流式细胞仪检测细胞凋亡情况。Hep2细胞接种于32只裸鼠,分组给予Lipo-ASODN进行治疗,2周后评价其抑瘤率。结果MTT实验显示Lipo-ASODN组浓度为1.0μmol/L和2.0μmol/L时细胞生长曲线逐渐降低,72h细胞抑制率可达52.5%和71.4%,两组间差异具有统计学意义(P=0.046),均显著高于对照组(P值分别为0.003和0.0004);转染48h后Lipo-ASODN组RT-PCR结果均可见生存素基因表达量明显少于对照组,Western Blot见生存素蛋白表达呈下降趋势;Lipo-ASODN组TUNEL检测可见细胞核内出现凋亡特征性阳性染色,流式细胞仪检测可见明显凋亡峰,而对照组无此现象。动物实验Lipo-ASODN组肿瘤抑制率可达48.1%和61.3%,显著高于对照组(P值分别为0.004和0.0006),且两组间差异具有统计学意义(P=0.032),呈一定的剂量依赖性。结论生存素反义寡核苷酸具有诱导喉癌细胞凋亡和体内抗肿瘤的作用,提示反义核酸技术可作为一种治疗手段应用于喉癌的基因治疗。更多还原

【Abstract】 Objective To observe the effect of survivin antisense oligodeoxynuclecotide (ASODN)on the apoptosis of human carcinoma of larynx cell line Hep2 and the inhibitory rate in nude mice model so as to discuss the selective blocking activity of antisense technique on gene expression seeking a new way for gene therapy of carcinoma of larynx. Methods Antisense oligodeoxynuclecotides survivin were transformed into human carcinoma of larynx cell line Hep2 by liposome Lipofectamine TM 2000. Within 72 h after transfection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay was used to detect cellular proliferation. Forty eight hours after transfection, reverse transcription-polymerase chain reaction (RT-PCR) assay was used to observe the expression of survivin gene, Western Blot assay for the protein, and terminal deoxynucleotide mediated nick end labeling(TUNEL) and flow cytometer for cellular apoptosis. Results Cellular inhibition rate of 72 h went up to 52.5% and 71.4% at 1.0 μmol/L and 2.0 μmol/L value in Lipo-ASODN groups which differed statistically remarkably(P=0.046),higher than that in controls in MTT assay(P=0.003 and 0.0004). Forty eight hours after transfection survivin gene expression in Lipo-ASODN groups were less than that in control group in RT-PCR assay. Survivin protein expression decreased in Western blot. In TUNEL assay, nuclear positive staining was observed and the apoptosis peak was observed in flow cytometer test, which were absent in controls. In nude mice of carcinoma of larynx model, the inhibitory rate in Lipo-ASODN groups got up to 48.1% and 61.3% higher than that of controls (P<0.004 and 0.0006), which differed remarkably(P=0.032) in a dose-dependently way. Conclusions The findings showed that the expression of survivin gene and protein induced cellular apoptosis in Hep2 cells after transfection of Lipo-ASODN and that the carcinoma of laryx in the nude mice model were inhibited by Lipo-ASODN which suggested that antisese technique can be an effective means in the gene therapy of carcinoma of larynx.更多还原