c-FLIP反义寡核苷酸诱导胚胎型横纹肌肉瘤细胞凋亡的研究

Study of c-FLIP-ASODN induced apoptosis in human rhabdomyosarcoma cells

目的探讨c-FLIP反义寡核苷酸(c-FLIPantisenseoligodeoxynucleotide,c-FLIP-ASODN)对c-FLIP的表达调控及诱导人胚胎型横纹肌肉瘤(rhabdomyosarcoma,RD)细胞凋亡的作用。方法以脂质体为载体,将c-FLIP-ASODN及对照序列分别转染RD细胞,分别通过WesternBlotting、RT-PCR方法检测ASODN对c-FLIP蛋白和mRNA表达量的调控,用流式细胞术测定ASODN干预前后RD细胞凋亡率的变化。结果与对照序列相比,Western印迹显示,转染了c-FLIP-ASODN的RD细胞其FLIPS和FLIPL的表达量分别下降为空白对照组的51%和57%;RT-PCR检测表明c-FLIP-ASODN能抑制c-FLIPmRNA的表达;诱导凋亡48h后,转染c-FLIP-ASODN的RD细胞凋亡率升高(29.3±6·3)%.结论c-FLIP-ASODN可有效转染RD细胞,下调c-FLIPmRNA表达,从而诱导RD细胞的凋亡,成为眼眶RD基因治疗的新靶向。

Objective To investigate the expressive regulation of c-FLIP antisense oligodeoxynucleotide （c-FLIP-ASODN）on c-FLIP and the effect of induced apoptosis in human rhabdomyosarcoma （RD） cells. Methods c-FLIP-ASODN and control sequence（Control-ODN） were transfected into RD cells with lipofectin, c-FLIP protein expression was examined by Western Blotting and the expression of c-FLIP mRNA was detected by RT-PCR. The apoptosis rate of RD cells transfected with c-FLIP-ASODN and Control-ODN before and after was assessed quantificationally by flow cytometric analysis. Results Compared with control sequence, Western Blotting showed that the expression level of c-FLIPs and c-FLIPL in RD cells were decreased to 51% and 57% by c-FLIP-ASODN; The expression of c-FLIP mRNA was detected by RT-PCR, it was inhabitive;After 48hours, compared with the untreated ones, the apoptosis rate of RD cells was increased to （29.3 ± 6.3） %. Conclusion c-FLIP-ASODN can effectively transfect RD cells and down-regulate c-FLIP mRNA expression so that it induce apoptosis in RD cells, c-FLIP-ASODN will become a new target of gene therapy for orbital RD.