反义寡核苷酸抑制大鼠胶质瘤多药耐药细胞株中Pgp相关基因表达的实验研究

目的通过研究反义寡核苷酸对大鼠胶质瘤多药耐药细胞株C6/adr中Pgp相关基因表达的抑制作用,探讨寡核苷酸在细胞内的代谢过程。方法根据MDR-1基因序列合成反义寡核苷酸转染胶质瘤耐药细胞,应用RT-PCR、流式细胞仪等方法,分别在转染后6、12、24、48h对其进行MDR-1mRNA水平及Pgp含量的检测。结果流式细胞及RT-PCR结果显示转染后6h即可发现胶质瘤耐药细胞Pgp和MDR-1mRNA减低,减低程度与转染后的检测时间相关,12~24h减低程度最为显著(P<0.05),48h后恢复至转染前的水平。结论反义寡核苷酸抑制大鼠胶质瘤细胞的Pgp表达和减低MDR-1mRNA水平具有明显的细胞内的代谢过程,了解其作用的规律性有助于选择适宜的用药时机,更大限度地提高疗效。

Experimental study of inhibiting process of gene expression of Pgp in multidrug resistant rat glioma cell line (C6/adr) by antisense oligodeoxynucleotides

Objective To explore the inhibiting process of gene expression of Pgp and metabolism of antisense oligodeoxynucleotides in multidrug resistant rat glioma cell line (C6/adr). Methods The antisense phosphorothioate-modified ODNs were designed to target MDR-1 gene and the ODNs were delivered by a cationic lipid vector to the rat glioma cells. The level of P-glycoprotein expression and MDR-1 mRNA of the treated glioma cells were detected by FCM and RT-PCR at the point of 6,12,24 and 48 hours after transfection. Results The outcome of FCM and RT-PCR showed that the glioma cells treated with antisense ODNs came into a decreased level of P-glycoprotein expression and MDR-1 mRNA,which began at 6 hours after transfection and came to a peak during 12 to 24 hours(P<0.05),and the level of pre-transfection was recovered after 48 hours. Conclusion There is an obviously inhibiting process of P-glycoprotein expression and the level of MDR-1 mRNA in rat glioma cells by antisense ODNs. Consequently, the proper time of chemical therapy can be chosen according to the rule of the antisense oligodeoxynucleotides metabolism .