【摘要】 目的探讨靶向survivin基因反义寡核苷酸(as ON)对MCF-7人乳腺癌细胞系凋亡及药物敏感性等生物学行为的影响。方法 MCF-7人乳腺癌细胞株分为正常组、脂质体组、正义组和反义组,正常组常规培养,并用Opti-MEM培养液代替转染液进行转染;脂质体组采用Lipofectamine 2000-Opti-MEM代替转染液进行转染;正义组采用survivin正义寡核苷酸进行转染;反义组采用survivin基因as ON转染。采用反转录聚合酶链反应检测各组survivin mRNA表达水平,四甲基偶氮唑盐法检测细胞抑制率;分别用紫杉醇处理MCF-7细胞株,并检测紫杉醇处理后survivin mRNA表达水平和细胞抑制率。结果 as ON转染后,MCF-7细胞中survivin mRNA表达水平显著降低,其中反义组survivin mRNA表达水平显著低于正常组、脂质体组和正义组(0.34±0.10比0.63±0.10、0.74±0.20、0.73±0.10),细胞抑制率显著高于正常组、脂质体组和正义组[(51.32±7.42)%比(3.72±0.10)%、(4.12±0.10)%、(4.53±0.10)%],差异有统计学意义(P<0.05)。转染后反义组细胞周期被阻滞于G2/M期;其中反义组G2/M期细胞比率显著高于正常组、脂质体组和正义组[(34.9±2.7)%比(21.5±2.8)%、(19.4±2.6)%、(18.8±1.9)%],反义组细胞凋亡率亦显著高于正常组、脂质体组和正义组[(16.3±1.2)%比(5.1±0.4)%、(5.3±0.5)%、(5.7±0.5)%],差异有统计学意义(P<0.05)。各组经紫杉醇处理后survivin mRNA表达水平和细胞抑制率均显著增加,与不加药组比较差异有统计学意义(P<0.05);其中反义组survivin mRNA表达水平显著低于正常组、脂质体组和正义组(0.62±0.15比1.37±0.33、1.47±0.33、1.33±0.26),细胞抑制率显著高于正常组、脂质体组和正义组[(84.75±9.62)%比(36.44±3.73)%、(41.45±5.36)%、(40.82±4.93)%],差异有统计学意义(P<0.05)。结论 survivin基因与MCF-7细胞增殖、抗凋亡、化疗耐药等生物学行为有关,通过as ON下调survivin基因的表达水平,能够促进肿瘤细胞凋亡和增强对化疗药物的敏感性,提示survivin基因是乳腺癌基因治疗的主要靶点之一。更多还原

【Abstract】 Objective To explore the effect of antisense oligonucleotide targeting survivin gene on biological behaviors of apoptosis and drug sensitivity of of MCF-7 human breast cancer cell line. Methods MCF-7 human breast cancer cell lines were divided into normal group,liposome group,sense group and antisense group: the normal group was cultured in Opti-MEM medium instead of the transfection solution; lipofectamine 2000-Opti-MEM was used in the liposome group,and the sense group was transfected with survivin oligonucleotide transfection,the anti-sense group was transfected with survivin antisense oligonucleotide( as ON). The expression level of survivin mRNA was detected by RT-PCR,and the inhibition rate was detected by MTT assay,MCF-7 cell lines were treated with taxol,and the expression level of survivin mRNA and cell inhibition rate were detected. Results After as ON transfection,the expression level of mRNA survivin in MCF-7 cells was significantly decreased,and the expression level of survivin mRNA in the antisense group( 0. 34 ± 0. 10) was significantly lower than that in the normal group( 0. 63 ± 0. 10),liposome group( 0. 74 ±0. 20),sense group( 0. 73 ± 0. 10)( P < 0. 05),and the inhibition rate( 51. 32 ± 7. 42) % was significantly higher than that in normal group( 3. 72 ± 0. 10) %,liposome group( 4. 12 ± 0. 10) %,sense group( 4. 53 ±0. 10) %( P < 0. 05). After transfection,the cell cycle was blocked at G2/ M phase,and the G2/ M phase cells rate in the anti-sense group( 34. 9 ± 2. 7) % was significantly higher than those in the normal group( 21. 5 ± 2. 8) %,liposome group( 19. 4 ± 2. 6) %,and sense group( 18. 8 ± 1. 9) %( P < 0. 05),and the apoptosis rate( 16. 3 ± 1. 2) % was significantly higher than that in the normal group( 5. 1 ± 0. 4) %,liposome group( 5. 3 ± 0. 5) %,and sense group( 5. 7 ± 0. 5) %( P < 0. 05). After treatment with taxol,survivin mRNA expression level and cell inhibition rate were significantly increased,and compared with no-medicine group,the difference had statistical significance( P < 0. 05); the survivin mRNA expression level of the antisense group( 0. 62 ± 0. 15) was significantly lower than that of the normal group( 1. 37 ± 0. 33),liposome group( 1. 47 ± 0. 33),and sense group( 1. 33 ± 0. 26); cell growth inhibition rate( 84. 75 ± 9. 62) % was significantly higher than that of the normal group( 36. 44 ± 3. 73) %,liposome group( 41. 45 ± 5. 36) %,and sense group( 40. 82 ± 4. 93) %( P < 0. 05). Conclusion Survivin gene is related to the biological behaviors of MCF-7 cell proliferation,anti-apoptosis and chemotherapy resistance,and the deregulation of expression level of survivin gene through as ON can promote tumor cell apoptosis and increase the sensitivity to chemical drugs,which suggests that survivin gene is one of the main targets for breast cancer treatment.更多还原