反义bcl-2 和反义survivin对人神经母细胞瘤细胞系SK-N-MC细胞生长的作用

目的研究反义bcl-2和反义survivin(svv)对人神经母细胞瘤(NB)细胞系SK-N-MC细胞生长的作用.方法利用基因重组技术,采用定向克隆构建在真核细胞中能够稳定表达反义bcl-2 或反义svv 的逆转录病毒载体PBabe puro-Asbcl-2和PBabe puro-Assvv,并分别经脂质体LipofectamineTM介导转染人NB细胞株SK-N-MC细胞,经嘌呤霉素(2.0 μg/ml)筛选得到抗性克隆,同时以空载体Pbabe puro 转染SK-N-MC作为对照;以免疫组化和Western 印迹分析反义bcl-2、反义svv对细胞内源性Bcl-2、SVV蛋白质的表达抑制作用,应用MTT法研究细胞(5×103 /孔)的生长和增殖情况,并接种于裸鼠(3只/组),观察107 个细胞在6周时的成瘤情况,以研究反义bcl-2、反义svv对SK-N-MC细胞生长的作用.结果反义bcl-2转染后的细胞SK-N-MC/PBabe puro-Asbcl-2中的Bcl-2表达明显降低,反义svv转染后的细胞SK-N-MC/PBabe puro-Assvv中的SVV表达也明显降低;且这两种细胞均生长缓慢, 裸鼠移植瘤体积均明显小于SK-N-MC原始细胞或转染空载体的细胞接种形成的移植瘤.结论稳定表达的反义bcl-2、反义svv均能够有效抑制SK-N-MC细胞内源性相应蛋白质Bcl-2、SVV的表达,提示这两个基因在NB细胞的肿瘤形成中均具有一定的作用.

Effects of antisense bcl-2 or survivin on the growth of human neuroblastoma cell line SK-N-MC

OBJECTIVE: To study the effects of antisense bcl-2 and survivin (svv) mRNA on the growth of human neuroblastoma (NB) cell line SK-N-MC cells. METHODS: The recombinant vectors PBabe puro-Asbcl-2 and PBabe puro-Assvv. were constructed by directed cloning of the EcoRI-BamHI fragments of bcl-2 cDNA or svv cDNA into the retroviral vector PBabe puro Human NB cell line SK-N-MC cells were transfected with PBabe puro-Asbcl-2, PBabe puro-Assvv, or blank vector PBabe puro as control by lipofectamine trade mark. The transfected cells were selected in the medium containing puromycine. The stably transfected cells were further studied for inhibition of protein expression of endogenous bcl-2 and SVV by immunohistochemical staining and Western blotting. The effect of antisense bcl-2 (Asbcl-2) and antisense svv (Assvv) mRNA on cell growth was determined by MTT method. The SK-N-MC cells transfected with the recombinant vectors were inoculated in nude mice to observe their carcinogenicity. RESULTS: Both the expression of bcl-2 and the expression of SVV significantly decreased in the antisense gene transfected cells in comparison to that in the original cells and cells transfected with blank vector. Seven days after the transfection, the MTT absorption (A(550)) was 0.374 +/- 0.001 5 in the cells transfected with Assvv, 0.289 +/- 0.000 8 in the cells transfected with Asbcl-2, both significantly lower than those in the original cells and cells transfected with blank vector (1.102 +/- 0.002 1 and 1.175 +/- 0.000 9 respectively). The induced tumors in the nude mice were smaller in the PBabe puro-Asbcl-2 transfected group and PBabe puro-Assvv transfected group than in the original and control groups. CONCLUSION: Stably expression of antisense bcl-2 and of antisense svv mRNA can effectively inhibit the expression of endogenous bcl-2 and SVV proteins. Both of them may play a role in the neoplastic formation of NB cells.