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为获得ZNRD1基因的编码区序列 ,构建反义真核表达载体 ,通过检测ZNRD1基因反义核酸转染对胃癌长春新碱耐药细胞SGC790 1/VCR阿霉素 (ADM)积蓄能力的影响 ,评价ZNRD1在抗胃癌细胞多药耐药性方面的应用前景。用PCR方法扩增目的基因序列 ,PCR产物经DNA序列测定证实后 ,采用分子克隆技术 ,将所获目的基因全长cDNA片段按反方向克隆入真核表达载体pcDNA3 1+ 的多克隆位点之间 ,对重组质粒进行酶切鉴定。用脂质体介导法将ZNRD1反义核酸转染SGC790 1/VCR ,流式细胞仪 (FACS)检测SGC790 1/VCR细胞内阿霉素的蓄积量。结果应用PCR反应扩增出特异性片段 ,经DNA序列分析 ,证实与文献报道的ZNRD1基因编码区序列一致 ;构建了反义真核表达载体pcDNA3 1 ZNRD1;转染SGC790 1/VCR细胞后 ,可提高细胞内ADM蓄积量。ZNRD1反义核酸转染后 ,胃癌多药耐药细胞株SGC790 1/VCR对抗肿瘤药物ADM的积蓄作用提高 ,提示ZNRD1反义核酸具有提高耐药细胞株细胞内药物浓度、恢复其药物敏感性、逆转多药耐药性的作用。
In order to obtain the coding sequence of ZNRD1 gene, an antisense eukaryotic expression vector was constructed. The effect of ZNRD1 antisense RNA transfection on the accumulation of doxorubicin (ADM) in vincristine-resistant gastric cancer cell line SGC790 1 / VCR was evaluated. Prospective Application of ZNRD1 in Multidrug Resistance of Gastric Cancer Cells. The PCR method was used to amplify the target gene sequence. After the PCR product was confirmed by DNA sequencing, the full-length cDNA fragment of the target gene was cloned into the multi-cloning site of the eukaryotic expression vector pcDNA3 1+ using molecular cloning technology Between the recombinant plasmid for restriction enzyme digestion. ZNRD1 antisense oligonucleotides were transfected into SGC790 1 / VCR by liposome-mediated method, and the accumulation of doxorubicin in SGC790 1 / VCR cells was detected by flow cytometry (FACS). Results The specific fragment was amplified by PCR and confirmed by DNA sequence analysis. The sequence of the coding region of ZNRD1 gene was confirmed. The antisense eukaryotic expression vector pcDNA3 1 ZNRD1 was constructed and transfected into SGC7901 / VCR cells Increase intracellular ADM accumulation. ZNRD1 antisense nucleic acid transfection, gastric cancer multidrug resistance cell line SGC790 1 / VCR on the accumulation of anti-tumor drug ADM increased, suggesting that ZNRD1 antisense nucleic acid drug-resistant cell lines to increase drug concentration, restore its drug sensitivity, Reversal of multidrug resistance.