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目的:研究脂质体介导的反义寡核苷酸cFLIP对人肺腺癌细胞系SPCA-1凋亡的影响,探讨反义技术选择性封闭目的基因表达进而发挥抗肿瘤作用的机制。方法:用脂质体LipofectamineTM2000将反义寡核苷酸cFLIP(Lipo-ASODN)和无义寡核苷酸cFLIP(Lipo-NASODN)片断导入人肺腺癌细胞系SPCA-1,通过MTT法分别检测细胞生长情况;应用RT-PCR检测cFLIP基因表达,并用WesternBlot检测cFLIP蛋白表达,同时原位末端标记(TUNEL)和流式细胞仪检测细胞凋亡情况。结果:MTT实验显示浓度为0.5μmol/L时Lipo-ASODN组细胞生长曲线逐渐降低,24h细胞抑制率可达71.52%,显著高于Lipo-NASODN组和Liposome对照组(P<0.01);转染24h后Lipo-ASODN组RT-PCR可见cFLIP基因表达量明显少于对照组(P<0.01),WesternBlot可见cFLIPS蛋白表达呈下降趋势,但cFLIPL变化不明显;TUNEL检测可见反义寡核苷酸组细胞核内出现阳性染色,流式细胞仪检测可见明显凋亡峰,且其作用呈一定的剂量依赖性。结论:反义寡核苷酸转染SPCA-1细胞后,cFLIPL/S基因与蛋白表达显著下调,表明此途径是cFLIPL/SmRNA基因片段诱导肿瘤细胞凋亡的机制之一。
OBJECTIVE: To study the effect of liposome-mediated antisense oligonucleotide cFLIP on the apoptosis of human lung adenocarcinoma cell line SPCA-1, and to explore the mechanism by which antisense technology can selectively block the target gene expression and exert its anti-tumor effect. Methods: LipofectamineTM2000 was used to introduce antisense oligonucleotide cFLIP (Lipo-ASODN) and non-sense oligonucleotide cFLIP (Lipo-NASODN) into human lung adenocarcinoma cell line SPCA-1 and detected by MTT assay The expression of cFLIP gene was detected by RT-PCR. The expression of cFLIP protein was detected by Western Blot. The cell apoptosis was detected by TUNEL and flow cytometry. Results: The MTT assay showed that the cell growth curve of Lipo-ASODN group decreased gradually at the concentration of 0.5 μmol / L, the inhibitory rate reached 71.52% at 24h, which was significantly higher than that of Lipo-NASODN group and Liposome control group (P <0.01) The expression of cFLIP gene in Lipo-ASODN group was significantly lower than that in control group after 24h (P <0.01). Western Blot showed that cFLIPS protein expression decreased, but cFLIPL did not change significantly; TUNEL assay showed that antisense oligonucleotide group Positive staining appeared in the nucleus, and obvious apoptosis peak was observed by flow cytometry, and the effect was dose-dependent. CONCLUSION: The expression of cFLIPL / S gene and protein is significantly down-regulated after antisense oligonucleotide transfection into SPCA-1 cells, indicating that this pathway is one of the mechanisms of cFLIPL / SmRNA gene-induced tumor cell apoptosis.