反义寡核苷酸逆转大鼠胶质瘤细胞多药耐药性的实验研究

目的探讨针对多药耐药基因(MDR-1)上特异位点的反义寡核苷酸对体外培养的大鼠胶质瘤细胞多药耐药性的逆转作用。方法根据MDR-1基因的碱基序列设计合成硫代修饰的寡核苷酸,通过阳离子脂质体介导转染胶质瘤耐药细胞,应用流式细胞仪、RT-PCR等方法,对转染后的耐药细胞进行P-170、MDR-1mRNA水平的检测;用MTT法对转染后的细胞进行化疗药物敏感性试验,评价寡核苷酸对多药耐药性的逆转效果。结果流式细胞结果显示转染后胶质瘤耐药细胞P-170表达明显低于转染前(P<0.05);RT-PCR可见转染后细胞的MDR-1mRNA水平减低;药物敏感性试验显示反义寡核苷酸转染后胶质瘤耐药细胞对化疗药物的敏感性增强(P<0.05),含有脂质体的转染体系组对化疗药物的敏感性显著高于不含脂质体的转染体系组(P<0.01)。结论特异序列的反义寡核苷酸能够明显抑制大鼠胶质瘤细胞的P-170表达和减低MDR-1mRNA水平,进而对胶质瘤细胞的多药耐药性产生明显的逆转作用;应用阳离子脂质体作为转染载体可显著提高转染效率。

Experimental study of inhibition of multidrug resistance of rat glioma cells (C6) by antisense oligodeoxynucleotides

Objective To explore the inhibition effect of antisense oligodeoxynucleotides(ODNs)on multidrug resistance of rat glioma cells(C6)in vitro.Methods The antisense phosphoroth ioate-modified ODNs were designed to target MDR-1gene and the ODNs were delivered by a cationic lipid vector to the rat glioma cells.The level of P-glycoprotein expression and MDR-1mRNA of the treated glioma cells were detected by FCM and RT-PCR.The drug sensitivity of the treated glioma cells were studied by MTT,and the reversal effect of antisense ODNs and sense ODNs were evaluated.The transfecting efficientcy of two kinds of transfection system were also evaluated.Results The out come of FCM and RT-PCR showed that the glioma cells treated by antisense ODNs came into a significantly inhibition of P-glycoprotein expression(P<0.05)and a decreased level of MDR-1mRNA.The results of multidrug resistance tests exhibited a significantly enhanced drug sensitivity in glioma cells after antisense ODNs transfection(P<0.05),and there was significant difference in drug sensitivity between with and without liposome (P<0.01).Conclusion The particular antisense ODNs can obviously inhibit P-glycoprotein expression and reduce the level of MDR-1mRNA in rat glioma cells.Sequentially,the antisense ODNs significantly reverse the multidrug resistance of the glioma cells.