硫代修饰bFGF寡核苷酸对B16细胞和L1210细胞增殖的影响

Influence on proliferation of B16 and L1210 cell lines by bFGF oligonuleotides with sulphate modification

目的研究bFGF寡核苷酸被硫代修饰后对肿瘤细胞增殖的影响。方法设计、合成bFGF寡核苷酸,用聚乙烯亚胺(jetPEI)介导bFGF寡核苷酸转染入黑色素瘤B16细胞和白血病L1210细胞,MTT法检测细胞增殖;微分干涉荧光显微镜观察硫代寡核苷酸在细胞中的定位;流式细胞仪分析寡核苷酸转染效率。结果正/反义硫代寡核苷酸被jetPEI介导成功转染入B16细胞和L1210细胞,在B16细胞均主要分布于细胞核,在L1210细胞分别主要分布于细胞核和细胞质。反义硫代寡核苷酸呈剂量依赖地抑制B16与L1210细胞增殖,且核苷酸突变能降低其对2种肿瘤细胞的增殖抑制。相应非修饰反义寡核苷酸也能显著抑制2种细胞增殖。正义硫代寡核苷酸能呈剂量依赖效应显著影响B16细胞增殖,显著高于非修饰正义寡核苷酸,且其抑制作用并未因核苷酸突变而降低。正义硫代寡核苷酸并不显著L1210细胞增殖,与非修饰正义寡核苷酸的效应基本一致。结论硫代修饰并未明显影响bFGF反义寡核苷酸在细胞内特异性结合核酸的性质,硫代反义寡核苷酸仍能以反义机制抑制肿瘤细胞增殖,但硫代修饰可能改变了bFGF正义寡核苷酸的某些性质,致使其于细胞内以一种非核酸特异性机制影响肿瘤细胞增殖。

[ Objective] To explore effects of bFGF oligonucleotides with sulphate modification on tumor cell proliferation. [Methods] bFGF oligonuleotides were transfected into melanoma B16 and lymphocytic leukaemia L1210 cell lines with jetPEI （polyethyleneimine）. The relative cell proliferation was from MTY analysis. Location of FITC-labeled phosphorothioate oligonucleotides was observed with fluorescence microscope, and positive rates of tumor cells transfected with antisense/sense phosphorothioate oligonucleotides were analyzed by Flow CytoMeter. [Results] bFGF antisense/sense phosphorothioate oligonuleotides were efficiently transfected into B16 and L1210 cells, which mainly located in nuclei of B16 cells, in cytoplasm and nuclei respectively in L1210 cells. Antisense phosphorothioate oligonuleotide inhibited the growths of B16 and L1210 cells in a dose-dependent manner, which were reduced for mismatch nucleotide significantly. The corresponding antisense phosphodiester oligonuleotide inhibited the proliferation of B16 and L1210 cells. Sense phosphorothioate oligonuleotide influenced the growth of B16 cells in a dose-dependent manner, more efficiently than sense phosphodiester oligonuleotide did. Mismatch nucleotide didn＇t reduce the effect of sense phosphorothioate ohgonuleotide on B16 cell line. Weak reducibility of L1210 cell proliferation by sense phosphorothioate oligonuleotide was consistent with that of sense phosphodiester oligonuleotide. [Conclusions]These findings suggested that phosphorothioate modification didn＇t significantly influence the intracellular quality of antisense oligonuleotide binding to nucleic acid and reducing tumor cell proliferation in a antisense manner, but sense phosphorothioate oligonuleotide biding to bFGF or other proteins influenced tumor cell proliferation.