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目的研究一段序列和胰岛素样生长因子2(IGF-2)基因第四启动子互补的特异性寡聚脱氧核苷酸(SODN)对Hep3B肝癌细胞株的抑制效应。方法根据IGF-2基因第四启动子序列人工合成一段与之互补的特异性脱氧核苷酸,并制备其长效的硫代衍生物,通过转染试剂将其导入高表达IGF-2的Hep3B 肝癌细胞株后,采用逆转录聚合酶链反应法和western blot技术分析SODN对IGF-2基因转录和蛋白表达的抑制效应,并通过四甲基偶氮唑盐法、流式细胞术、细胞克隆形成实验以及细胞侵袭和运动实验进一步观察SODN对Hep3B细胞的生长、细胞周期、体外克隆形成能力及侵袭和运动能力所产生的影响。结果空白对照组和导入非特异性寡聚脱氧核苷酸(CODN)对照组相比,导入SODN的Hep3B细胞,其IGF-2 mRNA 和蛋白的表达有明显降低;SODN对Hep3B细胞的增殖及运动能力不产生效应,但可以抑制其在培养板上的克隆形成数和穿透Matrigel的细胞数,抑制率分别为90.2%、95.5%。结论SODN具有“分子开关”作用,可以抑制和下调Hep3B肝癌细胞的IGF-2基因表达,并可逆转其部分恶性表型。
Objective To study the inhibitory effect of a specific sequence of oligodeoxynucleotide (SODN) complementary to the fourth promoter of insulin-like growth factor 2 (IGF-2) gene on Hep3B hepatocarcinoma cell line. Methods According to the fourth promoter sequence of IGF-2 gene, a specific deoxynucleotide complementary to it was synthesized and its long-acting thio derivative was prepared. The transfection reagent was used to introduce Hep3B The inhibitory effect of SODN on the transcription and protein expression of IGF-2 gene was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and western blot. The inhibitory effect of SODN on the expression of IGF-2 gene was analyzed by MTT, flow cytometry, Forming experiments and cell invasion and exercise experiments further observe the effects of SODN on Hep3B cell growth, cell cycle, in vitro clonality and invasion and exercise capacity. Results Compared with control group, the expression of IGF-2 mRNA and protein in Hep3B cells transfected with SODN was significantly lower in blank control group than that in control group. The proliferation and motor ability of Hep3B cells induced by SOD However, the number of clones and the number of cells penetrating Matrigel on the culture plates were inhibited with the inhibition rates of 90.2% and 95.5%, respectively. Conclusion SODN has the function of “molecular switch ”, which can inhibit and down-regulate IGF-2 gene expression in Hep3B hepatoma cells and reverse some of its malignant phenotypes.