CDK2干扰RNA对人脑胶质瘤细胞质蛋白质组的影响

Effects of CDK2 RNA interference on the cytoplasm proteome of SHG44 cells

目的构建人CDK2的干扰RNA真核表达载体,稳定转染人脑胶质细胞瘤SHG44细胞,经RT-PCR检测后,运用双向电泳-图像分析-质谱技术研究人脑胶质瘤细胞质蛋白质组的改变,探讨CDK2在SHG44细胞中的作用,为人脑胶质瘤的发生、发展的研究及诊断与治疗提供有价值的资料。方法 (1)构建CDK2干扰RNA真核表达载体并用双酶切和测序鉴定。(2)G418筛选阳性转染细胞克隆,制备稳定转染的SHG44细胞系。(3)通过RT-PCR比较转染后CDK2mRNA的表达量。(4)双向凝胶电泳-质谱技术-数据库搜索,比较转染前后细胞质蛋白质组的变化。结果 (1)成功构建了CDK2干扰RNA真核表达载体PGPU6/GFP/Neo-CDK2-1。(2)获得稳定转染细胞系,命名为PCDK2-1-SHG44细胞。(3)通过双向电泳-图像分析-质谱技术得到细胞质未见表达的蛋白质5个,包括醛缩酶A、波形蛋白等蛋白质。结论成功建立稳定转染CDK2干扰RNA的SHG44细胞系。质谱鉴定得到的这些差异表达的蛋白质涉及细胞的增殖、细胞凋亡的信号转导调节、正常细胞的恶性转化及发育调控以及肿瘤的发生、发展和耐药性形成。

Objective Eukaryotic expression vector of RNA interference specific for CDK2 that was stable transfectioned to inhibit the expression of CDK2 in SHG44 cell was constructed via the technique of RNA interference.The differentially expression of subcellular structure of the protein in SHG44 cell line was investigated by 2D-image master-MS and to offer valuable theoretical evidence for the occurrence and progress in glioma.Methods New eukaryotic expression vector of RNA interference specific for CDK2 were constructed and verified via dual enzyme cleavage and sequencing.Stable transfection cell line was obtained by G418 selection and to cultivate transfected SHG44 cell line.The mRNA contents of CDK2 was contrasted by RT-PCR.Differential expression proteins of cytoplasm were compared via MALDI-TOF-MS and searching of database.Results New targets of eukaryotic expression vector of RNA interference specific for CDK2 was constructed.Stable transfected cell line was constructed and named as PCDK2-1-SHG44.Five differential expression proteins in cytoplasm was identified by 2D-image master-MS.Conclusions SHG44 cell line was transfected by CDK2 siRNA.Differentially expressed proteins related to cell proliferation and apoptosis in regulation of signal transduction,malignant transformation of normal cells and tumor development,as well as the occurrence of regulation,development and the formation of drug resistance by mass spectrometry identification.