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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 the effect of survivin antisense oligonucleotide (ASODN) on the proliferation and apoptosis of human colon carcinoma cell line SW620 and to explore its molecular mechanism. METHODS: Designed to target the site of survivin binding to caspase-3, antisense oligonucleotides were synthesized and transfected into human colon carcinoma cell line SW620 by liposome. The inhibitory effect of survivin ASODN on the proliferation of SW620 cells was observed by MTT assay and the IC50 was determined. After transfection for 36 h, the nuclear changes of SW620 cells were detected by Hoechst33342 staining, and the expression of caspase-3 mRNA in SW620 cells by RT-PCR Expression and spectrophotometric assay of caspase-3 activity. Results: After 8h transfection, the yellow-green fluorescence was evenly distributed in SW620 cells. After treated with survivin ASODN at different concentrations for 44h, the proliferation of SW620 cells was significantly inhibited with IC50 of 1 × 10-6M. 2 × 10-7,4 × 10-7,6 × 10-7,8 × 10-7and1.2 × 10-6M survivin ASODN treatment, the cell growth inhibition rates were 15.38 ± 0.022%, 24.04 ± 0.023% , 30.87 ± 0.027%, 45.02 ± 0.018% and 65.01 ± 0.024%, respectively. Hoechest33342 stained fluorescence microscopy observed chromatin condensation and apoptotic bodies, detected by RT-PCR caspase-3mRNA expression, in addition caspase-3 activity was significantly increased. Conclusions Survivin ASODN, which is targeted to the site of survivin binding to caspase-3, can inhibit the proliferation and induce the apoptosis of human colon cancer cell line SW620. The mechanism is related to inducing the expression of caspase-3 and increasing the activity of caspase-3.