人脑胶质瘤CDK2干扰RNA新靶点的检验

Identification of new targets of human glioma CDK2 siRNA

目的构建4个新靶点CDK2干扰RNA真核表达载体,转染人脑胶质细胞瘤SHG44细胞,经逆转录-聚合酶链反应(RT-PCR)检测mRNA表达,获得干扰效果最好的真核表达载体,为CDK2成为人脑肿瘤标志物提供有价值的资料。方法 (1)构建4个新靶点CDK2干扰RNA真核表达载体并用双酶切和测序鉴定;(2)用脂质体法瞬时转染上述4个载体到SHG44细胞株;(3)通过RT-PCR比较转染后CDK2mRNA表达量,选出干扰效果最好的一个载体。结果 (1)成功构建了4个新靶点CDK2干扰RNA真核表达载体即pGPU6/GFP/Neo-CDK2-1、pGPU6/GFP/Neo-CDK2-2、pGPU6/GFP/Neo-CDK2-3、pGPU6/GFP/Neo-CDK2-4;(2)CDK2mRNA表达明显受抑制,并获得效果最好的CDK2干扰RNA真核表达载体。结论成功构建并筛选出效果最好的新靶点CDK2干扰RNA真核表达载体,使SHG44细胞的CDK2mRNA表达明显降低。

Objective To construct four new eukaryotic expression vectors of small interference RNA（siRNA） specific for CDK2 and confirm the interferential efficiency of siRNA on the expression of CDK2.Methods（1）Four new eukaryotic expression vectors of siRNA specific for CDK2 were constructed and identified by double enzymic digestion.（2）SHG44 cell line of human brain gliocytoma was transiently transfected with the four new vectors via oligofectamine.（3）Vector,with the strongest interferential efficiency,was confirmed by detecting the expression level of CDK2 mRNA using reverse transcription-polymerase chain reaction（RT-PCR）.Results（1）Four eukaryotic expression vectors of siRNA specific for new targets of CDK2 was constructed and denominated as pGPU6/GFP/Neo-CDK2-1,pGPU6/GFP/Neo-CDK2-2,pGPU6/GFP/Neo-CDK2-3 and pGPU6/GFP/Neo-CDK2-4.（2）The expression of CDK2 mRNA was obviously suppressed and the vector with the strongest interferential efficiency was obtained.Conclusion The eukaryotic expression vectors of siRNA,specific for new target of CDK2 and with the strongest interferential efficiency,was successfully constructed and indentified,which could obviously suppress the expression of CDK2 mRNA in SHG44 cell line.