Caspase-3在survivin反义寡核苷酸诱导人结肠癌细胞HT29凋亡时的表达

Expression of Caspase-3 in Survivin Antisense Oligodeoxy-nucleotid Induced Human Colorectal Carcinoma HT29 Cells Apoptosis

目的:靶向survivin基因中与caspase-3结合的部位合成反义寡核苷酸(ASODN)并将其转染至survivin表达阳性的HT29细胞中,观察HT29细胞中caspase-3的表达。方法:将survivin ASODN转染至HT29细胞中。噻唑蓝(MTT)法观察survivin ASODN的细胞毒作用,测定IC50;Hoechst 33342/PI双荧光染色,观察转染HT29细胞核变化;提取各组细胞总RNA,RT-PCR观察Caspase-3的表达。结果:不同浓度的survivin ASODN分别处理HT29细胞44h后,IC50值为1000nmol/L。1000nmol/Lsurvivin ASODN处理HT29细胞36h后,荧光显微镜下可见凋亡小体。PCR产物经过琼脂糖凝胶电泳,紫外透射仪下明显观察到500bp条带,产物经过序列测定证实为Caspase-3。结论:survivin ASODN可显著抑制HT29细胞增殖,诱导其凋亡。Caspase-3基因表达是其诱导HT29细胞凋亡的机制之一。

Objective：To investigate the expression of caspase-3 in the process of survivin antisense oligodeoxynucleotid （ASODN） induced HT29 cell apoptosis. Methods： ASODN targeting survivin gene were transferred into HT29 cells. IC50 was measured by MTr assay. The morphology changes of nucleons were observed by Hoechst 33342/PI staining. The expression of caspase-3 was measured by reverse transcription-polymerase chain reaction （RT-PCR）. Results： After HT29 cells treated with survivin ASODN for 44 hours, the IC50 was 1 000 nmol/L.After HT29 cells were treated with 1 000 nmol/L survivin ASODN for 36 hours, apoptosis bodies were observed by Hoechst 33342/PI staining. The agrose gel electrophoresis of PCR products and sequencing results showed the expression of Caspase-3. Conclusion： Survivin ASODN can effectively suppress the proliferation and induce apoptosis in human colorectal carcinoma HT29 cells and caspase-3 expression increasing may be one of the mechanisms.