ZNRD1基因的克隆及其反义核酸对胃癌耐药细胞阿霉素蓄积的影响

为获得ZNRD1基因的编友区序列，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SGC7901／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SGC7901／VCR，流式细胞仪（FACS）检测SGC7901／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,下实与文献报道的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SGC7901／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SGC7901／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。

CLONING OF ZNRD1 AND EFFECT OF ITS ANTISENSE VECTOR ON ACCUMULATION OF ADRIAMYCIN IN DRUG-RESISTANT GASTRIC CANCER CELLS

The aim of this study was to construct antisense of ZNRD1 encoding gene, and to transfect it into SGC7901/VCR cells, to seek measures to overcome multidrug resistance of gastric cancer cells. ZNRD1 cDNA amplified by PCR was confirmed by DNA sequencing, and then inserted into the multiple cloning site of the expressing vector pcDNA3 1 + with molecular cloning technique. The recombinant vector was identified by endonuclease digestion. Antisense recombinant vector was transfected into SGC7901/VCR cells using lipofectamine. Flow cytometric analysis was used to detect adriamycin (ADM) accumulation in SGC7901/VCR cells. The results showed that a fragment was obtained by PCR, and its sequence was consistent with ZNRD1 cDNA reported in the literature. After antisense recombinant vector having been transfected into SGC7901/VCR cell, ADM concentration in such cells was increased. The above results indicated that the antisense vector ZNRD1 could enhance the intracellular accumulation of ADM in SGC7901/VCR cells, which might be of potential treatment value.