Survivin反义寡核苷酸诱导SW620细胞凋亡过程中对caspase-3表达的影响

Impact of Caspase-3 Expressions during the Process of Induced Apoptosis in Human Colorectal Carcinoma SW620 Cells

目的:观察survivin反义寡核苷酸(ASODN)对人结肠癌细胞株SW620增殖、凋亡的影响,并初步探讨其分子机制。方法:靶向survivin基因中与caspase-3结合的部位设计、合成反义寡核苷酸并通过脂质体将其转染至人结肠癌细胞SW620中。噻唑蓝(MTT)法观察survivin ASODN对SW620细胞增殖的抑制作用,测定IC50;转染36h后,Hoechst33342染色荧光显微镜下观察检测SW620细胞核变化,RT-PCR检测survivin ASODN处理后SW620细胞中caspase-3mRNA表达,分光光度法检测caspase-3酶活性。结果:转染8h后,SW620细胞中可见黄绿色荧光均匀分布。不同浓度survivin ASODN处理SW620细胞44h后,SW620细胞增殖显著受到抑制,IC50为1×10-6M。2×10-7,4×10-7,6×10-7,8×10-7and1.2×10-6M的survivin ASODN处理后,细胞生长抑制率分别为15.38±0.022%、24.04±0.023%、30.87±0.027%、45.02±0.018%和65.01±0.024%。Hoechest33342染色后荧光显微镜下可观察到染色质凝集并出现凋亡小体,RT-PCR检测到caspase-3mRNA表达上调,另外caspase-3酶活性显著升高。结论靶向survivin基因中与caspase-3结合的部位设计合成的survivin ASODN可抑制显著结肠癌SW620增殖、诱导细胞凋亡,其机制与诱导caspase-3表达,提高caspase-3酶活性有关。

Objective： To observe of survivin antisense oligodeoxynucleotid （ASODN） on SW620 colon cancer cell lines proliferation, apoptosis and to explore its molecular mechanism. Methods： Targeting survivin gene in caspase-3 binding sites to design and synthesis of ASODN, different concentrations of survivin ASODN and control sequence （scrambled ODN） were transferred into SW620 cell lines by alipofectin reagent. The Thiazolyl blue （MTT） assay was used to measure the growth inhibitory rate and the IC50 was calculated. The morphologic changes in the nucleus were observed by Hoechst33342 staining. The mRNA expression of caspase-3 in SW620 cell lines were detected by retrotranscription polymerase chain reactio （RT-PCR）. Activity of caspase-3 enzyme was analyzed by spectrophotometry. Results： Eight hours after transtection, yellow-green fluorescence in the SW620 cells was uniformly distributed. SW620 cell lines growth were significantly inhibited after it was treated with different concentrations of survivin ASODN,IC50 were 1 ×10^--6M. The growth inhibitory with 2 ×10^-7,4 ×10^-7,6 ×10^-7,8 ×10^-7 and 1.2 ×10^-4 M of survivin ASODN was 15.38 ± 0.022%, 24.04± 0.023%, 30.87±0.027%, 45.02± 0.018% and 65.01 ± 0.024%. After 36h of survivin ASODN treatment, distinct morphologic changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were significantly by Hoechst33342 staining. The mRNA expressions of caspase-3 in SW620 cell lines treated with survivin ASODN were increased and caspase-3 acticity increased significantly. Conclusion： survivin ASODN can inhibit proliferation, induce apoptosis of SW620 cell lines, the mechanism may be related to inducting caspase-3 expression and then activating caspase-3.