四环素调控表达的反义bcl-2对人神经母细胞瘤细胞系SK-N-MC生长和凋亡的作用

Effects of tetracycline-controlled antisense bcl-2 expression on the growth and apoptosis of human neuroblastoma cell line SK-N-MC

目的 研究由四环素调控的反义bcl 2的表达对人神经母细胞瘤细胞系SK N MC细胞的生长和凋亡的作用并初步探讨其相关机制。方法 非定向克隆构建携带反义bcl 2cDNA片段的由四环素调控表达的真核细胞表达载体PUCCOMB1CMV/Asbcl 2 ;应用脂质体LipofectamineTM 介导转染SK N MC细胞 ,同时以空载体转染细胞作为对照。MTT法研究细胞的生长和增殖状况 ,提取DNA梯带和应用流式细胞术检测凋亡细胞。逆转录 聚合酶链反应 (RT PCR)研究细胞凋亡中bcl xL/bcl xS 基因在mRNA水平上表达的变化。Western杂交检测bcl 2及其上下游相关基因caspase 3、PARP的变化。结果 转染反义bcl 2的细胞生长增殖缓慢 ,在去血清培养的相同的时间内其凋亡细胞数也明显多于对照组细胞 ,提前出现DNA梯带。在细胞的凋亡过程中检测到非活性caspase 3酶原蛋白减少并检测到bcl 2、PARP蛋白的降解产物条带。但bcl xL/bcl xS 基因在mRNA水平上表达无明显改变。结论反义bcl 2mRNA可抑制SK N MC细胞的生长和增殖 ,促进去血清培养所诱导的SK N MC细胞的凋亡。在细胞凋亡过程中caspase 3被激活 ,bcl 2和PARP蛋白被裂解 ,但bcl xL/bcl xS 在反义bcl 2mRNA诱导的SK N MC细胞的凋亡过程中无变化。

Objective To study the effects of tetracycline-controlled antisense bcl-2 expression on the growth and apoptosis of human neuroblastoma cell line SK-N-MC and the related mechanisms. Methods The tetracycline-controlled antisense bcl-2 expressing vector PUCC OMB1 CMV /Asbcl-2 was constructed by inserting a 0.6 kb fragment antisense bcl-2 cDNA sequence into the plasmid PUCC OMB1 CMV . SK-N-MC cells were transfected with PUCC OMB1 CMV -Asbcl-2 or PUCC OMB1 CMV by Lipofectamine TM . The transfectant cells were further studied for growth viability and apoptosis induced by antisense bcl-2 expression controlled by tetracycline. The expression of bcl-x L/bcl-x S mRNA was examined by RT-PCR and the changes of expression of proteins caspase-3, bcl-2 and PARP were examined by Western-blot. Results The cell viability of the antisense bcl-2 transfected cells decreased significantly,and the apoptotic cells and DNA ladder could be found earlier in the antisense bcl-2 cells than in the empty vector transfected cells. We also found a decrease of non-activated caspase-3, cleavages of PARP and bcl-2 protein after treatment without fetal bovine serum (FBS) by Western-blot, but there was no change in the expression of bcl-x L/bcl-x S mRNA examined by RT-PCR during apoptosis. Conclusions The results suggested that tetracycline-controlled expression of antisense bcl-2 can effectively inhibit the cell viability of SK-N-MC cells, increase the sensitivity to apoptosis-inducing factor, as well as facilitate cell apoptosis after treatment of culture without FBS. Except bcl-x L/bcl-x S mRNA expression, activation of caspase-3, cleavage of protein PARP and bcl-2 were all associated with apoptosis.