反义核酸逆转Hep-2v细胞多药耐药的研究

The reversal of multidrug resistance in Hep-2v cell line by antisense oligodeoxynucleotides

目的 :探讨克服肿瘤细胞的多药耐药 (multidrugresistance ,MDR)的有效方法。方法 :用反义硫代磷酸寡聚脱氧核苷酸 (asODN)对Hep - 2v细胞进行多药耐药 (MDR)逆转实验 ,(1)将Hep - 2细胞经长春新碱(VCR)筛选建立Hep - 2v耐药细胞株为靶细胞 ;(2 )针对mdr1mRNA序列计算机辅助设计人工合成两个15mer反义寡聚脱氧核苷酸序列 :asODNⅠ、Ⅱ ,以硫代法修饰 ,其中序列Ⅰ和Ⅱ的作用位点相邻接 ,互补位点为MDR -mRNA的 330～ 35 9碱基 ;(3)MDR逆转实验 :两个反义序列分别或联合作用于Hep - 2v细胞 ,MTT法测定反义核酸作用前后VCR对Hep - 2v细胞IC50 ,比较其细胞毒作用 ;用流式细胞仪 (FCM )检测反义核酸作用后各实验组细胞表面糖蛋白P - 170的阳性率 ;RT -PCR法检测mdr1mRNA表达水平。结果 :两个反义核酸均使Hep - 2v细胞的P - 170阳性率下降 ,mdr1mRNA水平低调 ,VCR对Hep - 2v的细胞毒作用IC50 从90 0nmol L降至 <2 0 0nmol L。结论 :两个反义核酸用于Hep - 2v的MDR均获得明显的逆转效果 ,序列Ⅰ +Ⅱ联合作用的效果更强 。

Objective: To investigate the effectiveness of the antisense phosphorothioate oligonucleotides(asODN) in reversal of multidrug resistance in Hep-2v cell line. Methods: (1)The MDR cell line Hep-2v, selected by vincristine(VCR), was used as the target for this study. (2)Two 15mer antisense phosphorothoate oligodeoxynucleotides(asODN Ⅰ,Ⅱ) were designed against the mdr1-mRNA sequence. The two asODNs were tandem binding to bases from 330～359 of MDR1-mRNA. (3)The asODNs were administrated to the different group of the cultured Hep-2v cells. Quantitative analysis of P-170 was made by flow cytometry, and the level of mdr1-mRNA of the cells was detected by RT-PCR. The cellular toxicity of VCR was determined by MTT method.Results: The reversal of MDR was obtained effectively in the cell line after applied the asODNs. FCM indicated that P-170 was significantly reduced in the MDR cells. RT-PCR showed that the level of mdr1-mRNA in Hep-2v cells was down-regulated, and the IC 50 of VCR was decreased from about 900nmol/L to 200nmol/L. The more efficience was observed in the group of combined application of asODNⅠ and Ⅱ comparing with the use of asODN Ⅰor Ⅱ singly (P<0.05). Conclusions: Significantly reversal of MDR in Hep-2v cell line is obtained by using of the two asODNs. The more efficiency is observed in the test cell of combined application of asODNⅠand Ⅱ.It is suggested that the oligomers that are designed to bind in tandem to the target sites may have a synergic effect.