小鼠金属硫蛋白基因-I 在CHO细胞中稳定表达及其在医疗方面的应用

北京大学学报（自然科学版）

小鼠金属硫蛋白基因-I 在CHO细胞中稳定表达及其在医疗方面的应用

殷慎敏,李令媛,茹炳根

北京大学生命科学学院生物化学及分子生物学系，北京，100871

收稿日期:1996-01-29 出版日期:1996-07-20 发布日期:1996-07-20

Expression of Mouse Metallothioneins-ⅠGenomic Gene in CHO-dhfr^- Cells and Its Applications on Medical Therapy

YIN Shenmin, LI Lingyuan, RU Binggen

Department of Biochemistry and Molecular Biology, College of Life Sciences, Peking University, Beijing, 100871

Received:1996-01-29 Online:1996-07-20 Published:1996-07-20

摘要/Abstract

摘要： 将小鼠的MT-I基因插入pSV₂-dhfr质粒的EcoRI位点,构建了具有SV₄₀和MT双启动子并正向插入MT-I基因的表达载体。用改良的磷酸钙/DNA沉淀法将表达载体导入CHO-dhfr-细胞内，用不含次黄嘌呤(H)和胸腺嘧啶(T) 的选择培养基筛选出稳定表达MT阳性克隆D-22，经检测10⁶细胞的 MT 表达量约为1.8 μg，D-22细胞对Cd²⁺浓度抗性较之CHO-dhfr-细胞高14倍。为了研究金属硫蛋白对正常细胞所具有的保护作用，将不同浓度的顺铂分别处理无MT表达的CHO-dhfr-细胞和有MT表达的D-22细胞。实验发现顺铂对D-22细胞和CHO-dhfr^-细胞 50%生长抑制浓度(IC50)分别是0.145μmol/L 和0.04μmol/L。上述研究表明MT对正常细胞有一定的保护作用，因而通过诱导正常细胞提高MT的表达量，同时使用MT阻断剂阻断肿瘤组织的MT合成，这样可能会为治疗某些顽固肿瘤开辟一条新途径。

关键词: 金属硫蛋白(MT), CHO-dhfr-细胞, 细胞转染, 基因表达, 顺铂

Abstract: Mouse metallothioneins-I genomic gene was inserted into pSV2-dhfr plasmid at the sites of EcoRI and the recombinant plasmid was transferred into CHO-dhfr^-cells by DNA-calcium phosphate coprecitation method. Using western Blotting, RNA Hybridization, we could find the expression of MT-Ⅰat the level of 1.8μg/10⁶ cells in D-22 clonal cells which were selected by DMEM selective medium more than 8 weeks.The D-22 cells were treated with cisplatin and the IC50 to cispiatin was 0.145 μmol/L, being 3.1-fold resistance to cisplatin compared with the nontransfected cells—CHO-dhfr-cells (0.04 μmol/L). Theseresults provided the evidence of that Metallothionein may play an important role in protecting CHO cells from cytotoxicity of cisplatin. So increasing thesynthesis of MT by the inducer such as BSN and blocking the synthesis of MT bythe inhibitor such as proparglycine may be a potential way to treat some refractory carcinomas in chemotherapy.

Key words: Metallothionein (MT), CHO-dhfr^-Cells, Cell Transfection, Gene Expression, Cisplatin

中图分类号:

Q512.2

殷慎敏,李令媛,茹炳根 . 小鼠金属硫蛋白基因-I 在CHO细胞中稳定表达及其在医疗方面的应用[J]. 北京大学学报（自然科学版）.

YIN Shenmin,LI Lingyuan,RU Binggen. Expression of Mouse Metallothioneins-ⅠGenomic Gene in CHO-dhfr^- Cells and Its Applications on Medical Therapy[J]. Acta Scientiarum Naturalium Universitatis Pekinensis.

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https://xbna.pku.edu.cn/CN/Y1996/V32/I4/481

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