Cyclin D3正、反义表达质粒在淋巴瘤细胞系Raji中的转染与鉴定

目的 :构建人类cyclinD3正义和反义真核表达质粒 ,分别转染到淋巴瘤细胞系Raji细胞中 ,为探讨cyclinD3基因对淋巴瘤细胞生物学行为的影响提供研究基础。方法 :以Raji细胞总RNA为模板 ,通过RT -PCR获取cyclinD3全长cDNA ,克隆入 pGEM -T载体 ,再亚克隆到真核表达载体pcDNA3 .1中 ,构成含cyclinD3的表达质粒 pcD NA3 -cyclinD3。再运用DNA重组技术将cyclinD3基因反向克隆到pcDNA3 .1中 ,构建成cyclinD3反义表达质粒pcDNA3 -ascyclinD3。用脂质体介导的基因转染方法 ,将cyclinD3正、反义表达质粒和空质粒分别导入Raji细胞 ,经G418筛选建立稳定转染细胞株 ,用WesternBlot方法鉴定cyclinD3基因的表达。结果 :酶切鉴定和测序分析证实 ,实验成功构建了cyclinD3正、反义表达质粒。与转染cyclinD3正义表达质粒和空质粒的细胞相比 ,转染cyclinD3反义表达质粒的Raji细胞中cyclinD3表达水平下降。 结论 :正、反义cyclinD3在淋巴瘤细胞中的转染和表达 ,为进一步研究cyclinD3在淋巴瘤细胞增殖、细胞周期和细胞凋亡中的作用奠定了基础

Transfection and Establishement of Expression Plasmids of Sense and Antisense Cyclin D3 in Raji Cell Line

Objective:To construct expression plasmids of sense and antisense cyclin D3 gene and transfect them into Raji cell line to study the biological effects of cyclin D3 gene in lymphoma cells.Methods:Cyclin D3 cDNA was obtained by RT-PCR of total RNA extracted from Raji cells. The sequence was inserted into pGEM-T easy vector and then transfered into eukaryotic expression vector pcDNA3.1 to construct pcDNA3-cyclin D3. pcDNA3-ascyclin D3 plasmid was constructed by inserting cyclin D3 cDNA reversely to pcDNA3.1. The forward two recombinations and vector were then respectively transfected into Raji cell line by using liposome. The stable transfected cell lines were selected in medium containing geneticin(G418) and the expression level of cyclin D3 was certificated by Western Blot analysis.Results:It was comformed by restriction endonuclease digestion and DNA sequence analysis that the expression plasmid of sense and antisense cyclin D3 was successfully constructed. There was little change of cyclin D3 expression level between vector and sense cyclin D3 transfected cells, but the expression level of cyclin D3 in Raji cells transfected with antisense cyclin D3 was lower than that in vector transfected cells.Conclusions:The results of this study lay the foundation for further studying the role of cyclin D3 in cell cycle , cell proliferation and cell apoptosis of lymphoma.