CAJ | 학술논문

目的将表达特异性的小干扰RNA(siRNA)基因片段导入SMMC7721耐药细胞,干扰多药耐药相关蛋白1 (MRPl)基因后,观察其对细胞化疗敏感性的影响.方法使用浓度递增法制作耐阿霉素(ADM)细胞株,将ADM浓度在2 mg/L时培养出的细胞,命名为SMMC7721/ADM细胞,转染25、50、75 nmol/L siRNA沉默此耐药株MRP1基因,于转染后24、48、72 h使用实时定量逆转录聚合酶链反应(RT-qPCR)检测各组细胞MRPl mRNA表达水平.于12、24、36、48、60 h用噻唑蓝(MTT)法检测细胞对化疗药物的敏感性,加入反应试剂进行流式细胞仪检测细胞凋亡水平,使用Western blot分别检测细胞中MRP1蛋白表达含量.细胞注入裸鼠右侧肩胛区,14 d后测量肿瘤的长径和宽径.结果 SMMC7721/ADM细胞对阿霉素、5-氟尿嘧啶(5-Fu)、长春新碱(VCR)、奥沙利铂(OHP)的耐药系数分别为25.43、68.32、39.17、18.31.RT-qPCR检测沉默效率,可见MRP1的表达明显降低(P＜0.05).MRP1基因沉默后,Western blot技术可见MRP1蛋白明显降低.流式细胞仪可见细胞凋亡明显多于沉默前(P＜0.05).多药耐药性的检测可见细胞对上述药物耐药系数降低为5.73、15.86、2.49、1.84.动物实验监测肿瘤生长,可见肿瘤生长率明显降低.结论 SMMC7721耐药细胞株在使用siRNA沉默MRP1基因后,对化疗药的耐药性有明显下降.MRP1基因与SMMC7721肝癌细胞的多药耐药性显著相关,可通过沉默MRP1基因来提高耐药肿瘤细胞对化疗药物的敏感性.
목적 장표체특이성적소간우RNA(siRNA)기인편단도입SMMC7721내약세포,간우다약내약상관단백1 (MRPl)기인후,관찰기대세포화료민감성적영향.방법 사용농도체증법제작내아매소(ADM)세포주,장ADM농도재2 mg/L시배양출적세포,명명위SMMC7721/ADM세포,전염25、50、75 nmol/L siRNA침묵차내약주MRP1기인,우전염후24、48、72 h사용실시정량역전록취합매련반응(RT-qPCR)검측각조세포MRPl mRNA표체수평.우12、24、36、48、60 h용새서람(MTT)법검측세포대화료약물적민감성,가입반응시제진행류식세포의검측세포조망수평,사용Western blot분별검측세포중MRP1단백표체함량.세포주입라서우측견갑구,14 d후측량종류적장경화관경.결과 SMMC7721/ADM세포대아매소、5-불뇨밀정(5-Fu)、장춘신감(VCR)、오사리박(OHP)적내약계수분별위25.43、68.32、39.17、18.31.RT-qPCR검측침묵효솔,가견MRP1적표체명현강저(P＜0.05).MRP1기인침묵후,Western blot기술가견MRP1단백명현강저.류식세포의가견세포조망명현다우침묵전(P＜0.05).다약내약성적검측가견세포대상술약물내약계수강저위5.73、15.86、2.49、1.84.동물실험감측종류생장,가견종류생장솔명현강저.결론 SMMC7721내약세포주재사용siRNA침묵MRP1기인후,대화료약적내약성유명현하강.MRP1기인여SMMC7721간암세포적다약내약성현저상관,가통과침묵MRP1기인래제고내약종류세포대화료약물적민감성.
Objective To observe the effect of small interfering RNA (siRNA) targeting multidrug resistant-associated protein 1 (MRP1) gene in modulating multidrug drug resistance (MDR) and apoptosis of SMMC7721/ADM cells.Methods MDR was maintained by culturing the cells with 2 mg/L adriamycin (ADM) and MDR cells were named SMMC77212/ADM cells.The cells were transfected with 25,50 and 75 nmol/L siRNA using the reagent following the manufacturer' s protocol.MRP1 mRNA expression level was detected by using real-time reverse transcriptase-polymerase chain reaction (RT-qPCR)at 24,48 and 72 h.Total cells were harvested at 12,24,36,48 and 60 h after treatment with different drugs,then methyl thiazol tetrazolium (MTT) assay was used to determine drug sensitivity.Flow cytometry was employed to analyze cell cycle distribution,and the expression levels of proteins were analyzed by using Western blotting.In tumorigenicity experiment,SMMC-7721/ADM cells were transduced with or without MRP1 siRNA,and tumors were monitored for length and width at 14th day.Results MTT assay showed that the resistance index values of SMMC7721/ADM cells to ADM,5-Fu,VCR and OHP were 25.43,68.32,39.17 and 18.31,and 5.73,15.86,2.49 and 1.84 before and after treatment with siRNA respectively.RT-qPCR demonstrated the MRP1 mRNA expression was decreased significantly in SMMC7721/ADM cells after siRNA transfection.As compared with the expression of parental cells,MRP1 protein expression was apparently decreased,and flow cytometry showed the increased apoptosis after siRNA transfection.Conclusion Inhibition of MRP1 by siRNA enhanced the selectively restored sensitivity to drugs.The MRP1 siRNA might represent a new therapeutic option for hepatocellular carcinoma.