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１．８μｇ，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。
Mouse metallothioneins I genomic gene was inserted into pSV 2 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/10 6 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 IC 50 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). These results provided the evidence of that Metallothionein may play an important role in protecting CHO cells from cytotoxicity of cisplatin. So increasing the synthesis of MT by the inducer such as BSN and blocking the synthesis of MT by the inhibitor such as proparglycine may be a potential way to treat some refractory carcinomas in chemotherapy.
出处
《北京大学学报（自然科学版）》
CSCD
北大核心
1996年第4期481-487,共7页
Acta Scientiarum Naturalium Universitatis Pekinensis
基金
国家863生物高技术项目资助
关键词
金属硫蛋白
细胞转染
基因表达
CHO细胞
医疗
Metallothionein (MT)
CHO dhfr -Cells
Cell Transfection
Gene Expression
Cisplatin
