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目的探讨稳定转染CDK2干扰RNA对人肝癌细胞株HepG2细胞生物活性及细胞核蛋白质的改变。方法构建稳定转染PGenesil-1-CDK2的HepG2细胞系,MTT法检测细胞增殖、流式细胞术检测细胞周期的改变。通过RT-PCR和双向凝胶电泳-质谱技术-数据库搜索,比较转染前后CDK2 mRNA的表达和细胞核蛋白质的变化。并通过Western blot法对显著差异蛋白进行验证。结果与空质粒组PHK-siRNA-HepG2细胞和未转染组HepG2细胞相比,PCDK2-siRNA-HepG2组细胞的生长速度减慢(P<0.01),稳定转染CDK2 RNAi组细胞的CDK2 mRNA表达水平显著下降。通过双向电泳-质谱技术得到4个稳定转染CDK2 siR-NA的HepG2细胞不表达的蛋白质,Western blot法证实双向电泳结果的可信性。结论 CDK2干扰RNA可明显降低HepG2细胞CDK2 mRNA的表达,抑制HepG2细胞的增殖,干扰后的HepG2细胞不表达的蛋白质分别是类核糖体蛋白S12、β-肌动蛋白、锌指蛋白276和伴侣蛋白10相关蛋白。
Objective To investigate the changes of biological activity and nuclear proteins in human hepatocellular carcinoma HepG2 cells stably transfected with CDK2 interfering RNA. Methods HepG2 cells stably transfected with PGenesil-1-CDK2 were constructed and cell proliferation was detected by MTT assay. Cell cycle was detected by flow cytometry. The expression of CDK2 mRNA and the changes of nuclear proteins were compared by RT-PCR and two-dimensional gel electrophoresis-mass spectrometry-database search. Western blot was used to verify the significant proteins. Results The growth of PCDK2-siRNA-HepG2 cells was slower than that of untreated cells in PHK-siRNA-HepG2 cells (P <0.01). The expression of CDK2 mRNA in cells stably transfected with CDK2 RNAi The level dropped significantly. Four proteins that were not expressed in HepG2 cells stably transfected with CDK2 siR-NA were obtained by two-dimensional electrophoresis-mass spectrometry. The result of two-dimensional electrophoresis was confirmed by Western blot. Conclusion CDK2 interference RNA can significantly reduce the expression of CDK2 mRNA in HepG2 cells and inhibit the proliferation of HepG2 cells. The interfering HepG2 cells do not express proteins like ribosomal protein S12, β-actin, zinc finger protein 276 and chaperonin 10 related proteins.