MGMT基因导入对真核细胞保护作用的实验研究

The protective effect on eukaryotic cells transferred with MGMT gene

目的克隆六氧甲基鸟嘌呤-DNA-甲基转移酶(MGMT)基因并构建表达载体,导入真核细胞表达,研究MGMT对真核细胞保护作用。方法利用RT-PCR克隆人肝细胞MGMT基因并重组构建真核表达载体。脂质体法导入K562细胞及人外周血单个核细胞(PBMNC),用实时定量PCR法检测目的基因的表达。MTT法检测转基因细胞对烷化剂耐药性的改变。结果成功克隆人MGMT基因,转染K562细胞和PBMNC后,转基因组MGMT mRNA表达水平分别是转染空载体组的13．4倍(P<0．01)和4倍(P<0．01)。Western blot结果显示MGMT蛋白表达在转基因组中明显高于转空载体组及对照组。耐药性实验结果显示目的基因导入后,K562稳定转染细胞和PBMNC瞬时转染细胞对烷化剂的半数抑制浓度分别提高到原来的7倍和2倍。结论MGMT基因导入可使真核细胞对烷化剂的耐药性得到不同程度的增强,从而对细胞起到保护作用。

Objective To clone 60-methylguanine-DNA- methyltransferase （MGMT） gene and construct an expression vector to study the protective effect on eukaryotic cells transferred with MGMT gene. Methods Mammalian expression vector containing MGMT gene cloned from human hepatocytes by RT-PCR was constructed and transferred into K562 cells and human peripheral blood mononuclear cells （PBMNCs） via liposome. 3-（4,5-dimethylthiazol-2-yl）-2,5-dipheyltetrazolium bromide （MTF）was used to detect the drugresistance of the MGMT transfected cells. Results MGMT was successfully cloned . Realtime PCR showed the mRNA expression in the gene-transfected K562 cells and PBMNCs were 13.4 times（ P 〈 0.01 ） and 4 times （P 〈0.01 ） that in the empty vector-transfected controls. Results of Western blot showed significantly higher expression of MGMT in gene-transfected group than in other two groups. The IC50 values were increased to 7 times and 2 times that of the originals respectively in stable-transfected K562 cells and transient-transfected PBMNC. Conclusion The alkylating agent-resistance of eukaryotic cells transfected with MGMT was promoted to higher levels.