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目的 Survivin是一种凋亡抑制蛋白(inhibitorofapoptosisproteins ,IAP)家族成员,在几乎所有肿瘤组织中特异性表达,而在正常成年终末分化组织中低表达甚至不表达。选择五组小鼠survivin反义寡核苷酸,并从中进步筛选效果显著的反义寡核苷酸,在小鼠体内进一步验证其抑制肿瘤生长的能力。方法 设计合成特异性靶向生存素的反义寡核苷酸五组。利用脂质体转染技术将其转入肺癌细胞D12 1,Westernblot检测survivin的表达抑制效应,从中筛选效果显著的反义寡核苷酸,确定最佳浓度,将此浓度下转染的D12 1细胞经小鼠尾静脉注入,观察生存曲线。结果 各组反义寡核苷酸在4 0 0nmol·L-1转染细胞,Westernblot结果显示NO .5组survivin表达程度减弱,选取NO .5反义寡核苷酸,在不同浓度下观测转染2 4h后,细胞凋亡情况及survivin表达状况。2 0 0nmol·L-1时,sur vivin表达出现减弱,但细胞生长尚未受到影响;4 0 0nmol·L-1时细胞开始出现凋亡,指数明显高于对照组(P <0 .0 5 ) ,以6 0 0nmol·L-1转染组最为明显。用NO .5反义寡核苷酸在2 0 0nmol·L-1浓度下转染D 12 1细胞,将此细胞致瘤小鼠,小鼠的生存期明显延长。结论 因为survivin基因在多数恶性肿瘤组织中丰富表达,选择合适的sur vivin反义寡核苷酸,将有利于应用在肿瘤的
Purpose Survivin is a member of the inhibitor of apoptosis protein (IAP) family, which is specifically expressed in almost all tumor tissues, but not expressed in normal adult terminal differentiated tissues. Five groups of mouse survivin antisense oligonucleotides were selected and the antisense oligonucleotides with significant effect were screened out to further verify their ability to inhibit tumor growth in mice. Methods Five sets of antisense oligonucleotides targeting specific survivin were designed. The recombinant plasmid was transfected into lung cancer cell line D12 1 by lipofection technique. The expression of survivin was detected by Western blot. The antisense oligodeoxynucleotides with significant effect were screened out to determine the optimal concentration. The D12 1 The cells were injected through the tail vein of mice to observe the survival curve. Results The antisense oligonucleotides in each group were transfected into the cells at 400 nmol·L-1. The results of Western blot showed that the expression of survivin in NO. 5 group was weakened. NO. 5 antisense oligonucleotide was selected and observed under different concentrations Twenty-four hours after dyeing, apoptosis and survivin expression. At 200 nmol·L-1, the expression of Sur-vivin decreased but the cell growth was not affected. The apoptosis began to occur at 400 nmol·L-1, which was significantly higher than that of the control group (P <0.05) , Which was the most obvious at 600 nmol·L-1 transfection. Using NO. 5 antisense oligonucleotide to transfect D 12 1 cells at the concentration of 200 nmol·L-1, the survival time of tumor-bearing mice and mice was significantly prolonged. Conclusion Because survivin gene is abundantly expressed in most malignant tumors, the selection of suitable sur-vivin antisense oligonucleotides will be of benefit in tumor application