Ss-A/Ro核蛋白60 ku亚单位在胃癌多药耐药中的作用

Function of Ss-A/Ro ribonucleoprotein 60-ku subunit in multidrug resistance of gastric cancer

目的:对Ro 60在胃癌多药耐药中的功能进行研究．方法:克隆Ro 60编码基因,构建Ro 60编码基因的正义真核表达载体,将其转导入SGC7901 细胞,应用半定量RT-PCR技术,对基因转染细胞进行鉴定,通过MTT法进行体外药物敏感性分析,借助流式细胞仪检测细胞内蓄积的阿霉素．结果:成功构建了Ro 60正义真核表达载体, 应用脂质体介导法将其转导入SGC7901,Ro 60真核表达载体转染SGC7901细胞后其表达量明显增加,体外药物敏感性试验提示其对长春新碱、5-氟尿嘧啶、丝裂霉素、顺铂、阿霉素的敏感性减低,SGC7901细胞的IC50值 (mg/L)与未转染细胞和转染空白载体细胞相比,显著增加(2．28±0．11 vs 0．45±0．04,0．65 ±0．05;2．89±0．14 vs 0．61±0．21,0．90±0．11; 1．92±0．03 vs 0．54±0．03,0．75±0．21;1．41± 0．06 vs 0．45±0．03,0．54±0．03;0．28±0．03 vs 0．14±0．01,0．14±0．01;均P<0．01),细胞内阿霉素蓄积显著减少(56．30±2．49 mg/L vs 92．83 ±3．63,87．38±2．94 mg/L,P<0．01)．结论:Ro 60真核表达载体转染SGC7901细胞后显示了一些多药耐药的特性,提示Ro 60在胃癌多药耐药中发挥了一定的作用．

AIM： To investigate the possible function of Ss-A/Ro ribonucleoprotein 60-ku subunit （Ro 60） in the multidrug resistance （MDR） of gastric cancer cell line. METHODS： The Ro 60 encoding gene was cloned using reverse transcription polymerase chain reaction （RT-PCR）. DNA recombination technique was used to construct Ro 60 sense eukaryotic expression vector, and then the product was transfected into SGC7901 cells by Lipofectamine^TM 2000. Drug susceptibility in the chemotherapy was analyzed by MTT assay, and IC50 value of gastric cancer cells to chemotherapy drugs was calculated. The intracellular accumulation of adriamycin in gastric cancer cells was measured using fluorescence-activated cell sorting. RESULTS： The expression level of Ro 60 in SGC7901 was increased after transfection with sense genes. In vitro drug sensitivity assay showed that SGC7901 cells transfected with Ro 60 genes significantly decreased sensitivity to vincristine （IC50：2.28 ± 0.11 mg/L vs 0.45 ± 0.04, 0.65 ± 0.05 mg/L, P 〈 0.01）, 5-fluorouracfl （5-FU） （IC50：2.89 ± 0.14 mg/L vs 0.61 ± 0.21, 0.90 ± 0.11 mg/L, P 〈 0.01）, mitomycin （IC50：1.92 ± 0.03 mg/L vs 0.54 ± 0.03, 0.75 ± 0.21 mg/L, P 〈 0.01）, cisplatin （IC50：1.41 ± 0.06 mg/L vs 0.45 ± 0.03, 0.54 ± 0.03 mg/L, P 〈 0.01） and adriamycin （IC50： 0.28 ± 0.03 mg/L vs 0.14 ± 0.01, 0.14 ± 0.01 mg/L, P 〈 0.01）, when compared with SGC7901 and SGC7901-pcDNA3.1 cells. The intracellular accumulation of adriamycin in the cells transfected with Ro 60 gene was markedly decreased in comparison with that in SGC7901 and SGC7901-pcDNA3.1 cells （56.30 ± 2.49 mg/L vs 92.83 ± 3.63, 87.38 ± 2.94 mg/L, P 〈 0.01）. CONCLUSION： SGC7901 cells transfected with Ro 60 sense genes show some competence of MDR. Ro 60 may play a certain role in the MDR of gastric cancer.