摘要
目的采用RNA干扰技术阻断Survivin基因的表达,观察其诱导乳腺癌细胞凋亡及对表阿霉素的化疗增敏作用。方法构建靶向Survivin的小干扰RNA(siRNA)真核表达载体,采用lipofectamineTM2000转染乳腺癌细胞MCF-7,采用半定量RT-PCR、免疫组化技术检测转染前后MCF-7细胞Survivin基因表达的变化;采用TUNEL法检测其诱导MCF-7细胞凋亡及对表阿霉素的化疗增敏作用。结果靶向Survivin的序列特异性的siRNA可以有效地抑制MCF-7细胞Survivin基因的表达,在mRNA水平其表达抑制率为64.91%;在蛋白质水平其表达抑制率为79.72%;转染靶向Survivin的siRNA真核表达载体48h后可以诱导8.75%的细胞凋亡;阻断Survivin基因的表达,可以显著提高MCF-7细胞对表阿霉素的敏感性,靶向Survivin的siRNA联合表阿霉素可以诱导24.21%的细胞凋亡。结论本研究所构建的靶向Survivin的siRNA真核表达载体可以有效、特异地阻断Survivin基因的表达,阻断Survivin基因的表达可以诱导一定程度的MCF-7细胞自发凋亡,并显著增强其对表阿霉素的敏感性。
Objective To use the sequence-specific siRNA knocking down the expressions of Survivin gene and inducing breast cancer MCF-7 cell line to apoptosis, and to couple the siRNA with Survivin for investigating the effects of MCF-7 cell induced to apoptosis and the chemotherapy sensitivity of breast cancer cell treated to epirubiein. Methods The molecular cloning technique was applied to construct the eukaryotic expression vector of siRNA against Survivin, and lipofectamine^TM2000 was used to transfect MCF-7 cell. Survivin expressions were detected by semi-quantitive RT-PCR and immunohistochemical SABC methods. The effects of inducing MCF-7 cell apoptosis and enhanced chemotherapy sensitivity to epirubicin were assessed by TUNEL method. Results The sequence-specific siRNA can, effectively and specifically, knock the expressions of Survivin gene down at both mRNA and protein levels, in which the expression inhibition rates were 64.91% and 79.72% respectively. After 48 h, 8. 75 % cells transfected with siRNA expression vector were induced to apoptosis; Coupling siRNA against Survivin with epirubicin can induce the cell apoptosis rate up to 24.21%. Conclusions In the study, the siRNA against Survivin can, effectively and specifically, decrease the expressions of Survivin gene in MCF-7 cell; blocking the expressions of Survivin can, in certain degree, induce MCF-7 cell to apoptosis and enhance cell chemotherapy sensitivity to epirubicin significantly;Survivin RNAi has a great potential value in the gene therapy of breast cancer.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2006年第2期221-225,共5页
Journal of Sichuan University(Medical Sciences)
基金
陕西省科技攻关项目(批准号2003K10-G35)资助
