硫酸镉对FL/P4501A1细胞中人肺癌易感基因P4501A1的影响

The effects of CdSO 4 on human lung cancer susceptible gene P450 1A1 in FL/P450 1A1 cell

目的 观察重金属化合物硫酸镉 (CdSO4 )在体外对人羊膜FL/P45 0 1A1细胞内P45 01A1DNA序列的改变作用 ,了解镉化合物对肺癌易感基因的影响。方法 利用不同剂量的CdSO4 处理FL/P45 0 1A1细胞 ,其半数抑制浓度 (IC50 )为 2 4.5 5mg/ml。以 1/ 4IC50 终浓度CdSO4 处理FL/P45 0 1A1细胞 48h后提取细胞pREP 9/P45 0 1A1质粒。由于 pREP 9缺少通用序列 ,BamHI酶切 ,低熔点琼脂糖电泳分离P45 0 1A1cDNA片段。BamHI酶切 pGEM～ 3Zf(+ ) ,CIP处理 ,并与 2倍摩尔P45 0 1A1cDNA片段混合 ,乙醇沉淀 ,沉淀物加入 18μlTE ,加 2 μl 10倍连接酶缓冲液 ,混匀 ,加10UT4DNA连接酶 ,混匀 ,12℃保温 16～ 2 0h。凝胶电泳分析连接结果。鉴定合格连接物转化DH5α菌 ,X gal喷洒对重组子进行蓝 /白颜色筛选。选择含重组子的宿主菌进行P45 0 1A1cDNA测序。结果 在体外 ,CdSO4 未引起FL/P45 0 1A1cDNA序列改变。结论 CdSO4 对FL/P45 0 1A1细胞中肺癌易感基因P45 0

Objective To study the effects of CdSO 4 on lung cancer susceptible gene by observing the changes on the P450 1A1 DNA sequence in FL/P450 1A1 cells in vitro.Methods Human amnion FL/P450 1A1 cells were reacted with various doses of CdSO 4,its 50% inhibitory concentration(IC 50 ) was 24.55 mg/ml. pREP 9/P450 1A1 plasmids were cut with BamHI and P450 1A1 cDNA fragments were isolated by low melting point agarose gel electrophoresis.pGEM～3Zf(+) plasmids were cut with BamHI and were treated by CIP,mixed with P450 1A1 cDNA fragments and were precipitated by ethanol.TE,buffer and T4 DNA ligase were added into the mixtures and incubated at 12 ℃ for 16～20 h.The results were analysed by agarose gel electrophoresis.The elected recombinants infected DH5α bacteria,recombinants were screened by X gal.P450 1A1 cDNA were sequenced from DH5α/P450 1A1 bacteria. Results P450 1A1 cDNA sequence was not changed by CdSO 4. Conclusion No effects of cadium compounds on human lung cancer susceptible gene P450 1A1 in vitro were found.