人核糖体蛋白S13与胃癌细胞多药耐药性的实验研究  

Ribosomal protein S13 related multidrug resistance in human gastric cancer

作　　者：翟惠虹[1] 郭新宁[2] 时永全[1] 胡建国 杨力 王新[1] 兰梅[1] 樊代明[1] 

机构地区：[1]第四军医大学西京医院消化病研究所,西安现在710032 [2]宁夏医学院附属医院消化内科

出　　处：《中华消化杂志》2004年第3期139-142,共4页Chinese Journal of Digestion

基　　金：国家自然科学重点基金 ( 3 0 0 3 0 14 0 )

摘　　要：目的　探讨人核糖体蛋白S13(RPS13)在胃癌多药耐药 (MDR)机制中的作用。方法　采用RT PCR法扩增RPS13cDNA片段编码区序列全长 ,DNA重组技术构建正反义真核表达载体 ,经脂质体介导转染胃癌细胞SGC790 1及胃癌耐药细胞SGC790 1/VCR ,斑点杂交检测转染细胞mRNA水平的变化 ;MTT法测定细胞对化疗药物的敏感性 ,流式细胞仪检测细胞周期。结果　RT PCR法成功扩增出RPS13cDNA片段编码区序列全长 ,并构建正反义真核表达载体 ;斑点杂交试验证实 :正义转染细胞RPS13mRNA水平上调 ,反义转染细胞其mRNA水平下调。RPS13正义核酸转染SGC790 1细胞后 ,细胞对阿霉素、5 氟尿嘧啶和长春新碱的敏感性降低 ;转染反义核酸后 ,耐药细胞对丝裂霉素和长春新碱的敏感性增加。细胞周期测定表明高表达RPS13后 ,G1期、S期和G2期细胞的比例分别为 4 7.0 %、33.2 %和 19.8% ;低表达RPS13后 ,G1期、S期和G2期细胞的比例分别为 6 2 .9%、1.0 %和 36 .1%。结论　RPS13参与胃癌耐药细胞SGC790 1/VCR的多药耐药。Objective To study the effect of ribosomal protein S13(RPS13) encoding genes on the development of mutidrug resistance (MDR) in human gastric cancer cell line. Methods RPS13 cDNA was amplified by RT-PCR. The sense and antisense eukaryotic expression vectors were constructed by DNA recombination. Gastric cancer cell line SGC7901 and Vincristine-resistant SGC7901/VCR cells were transfected with the sense and antisense recombinant vectors respectively using liposome-mediated method. RNA dot blotting assay was used to verify the changes of mRNA level in stable clones. To investigate effects of the sense, antisense vector transfection on the chemotherapeutic drug sensitivity, thiazolyl blue (MTT) cytotoxicity assay was used. Cell cycle was detected by flow cytometry (FCM). Results Whole length of RPS13 cDNA gene was amplified by RT-PCR. The sense, antisense eukaryotic expression vectors were constructed by the directed cloning of the target genes into eukaryotic expression vector pcDNA 3.1(+). RNA dot blotting assay suggested that mRNA level of the RPS13 was up-regulated in the sense recombinant vector transfected cells, and down-regulated in the antisense recombinant vector transfected cells. By MTT cytotoxicity assay, the enhanced resistance to adriamycin, 5-fluorouracil and vincristine was found in the RPS13 sense recombinant vector transfected SGC7901 cells. RPS13 antisense recombinant vector rendered SGC7901/VCR cells partially sensitive to mitomycin and vincristine. Cell cycle analysis suggested that the proportion of G1, S, and G2 cells was 47.0%, 33.2% and 19.8% respectively in up-regulated RPS13 cells; the proportion of G1, S and G2 cells was 62.9%, 1.0% and 36.1% respectively in down-regulated RPS13 cells. Conclusions RPS13 may take part in the mediation of MDR in gastric cancer cells.

关 键 词：人核糖体蛋白S13 胃癌 多药耐药性 RT-PCR法 脂质体 基因转染 克隆 

分 类 号：R735.2[医药卫生—肿瘤]