小鼠p27~(kip1)真核表达载体的构建及其对单个核细胞周期的影响

目的构建小鼠p27kip1真核表达载体,探讨其对单个核细胞生长情况和细胞周期的影响,为进一步研究p27kip1在疾病防治中的作用奠定基础。方法将小鼠p27kip1基因克隆至pcDNA3.0真核表达载体上,并经脂质体介导转染至小鼠单个核细胞中,应用MTT法和流式细胞仪分析转染细胞生长情况和细胞周期。结果经酶切分析和测序鉴定,成功构建了小鼠pcDNA3.0/p27kip1重组质粒;经pcDNA3.0/p27kip1转染的单个核细胞中p27蛋白表达增多,其生长增殖不仅受到明显抑制,而且处于G1/G0期的细胞明显增加,处于S期的细胞明显减少。结论构建的pcDNA3.0/p27kip1真核表达载体能够在单个核细胞中表达,且高表达的pcD-NA3.0/p27kip1可以通过抑制细胞周期的方式抑制单个核细胞的生长。

Construction of p27kip1 eukaryotic expression plasmid to investigate its influence on mononuclear cell cycle

Aim To construct p27kip1 eukaryotic expression plasmid to investigate its influence on mononuclear cell proliferation and cell cycle for further researching the role of p27kip1 in disease prevention and treatment.Methods p27kip1 gene was cloned into pcDNA3.0 eukaryotic expression,then pcDNA3.0/p27kip1 mediated by liposome was transfected into mononuclear cell.MTT assay and flow cytometry were used to analyze cell proliferation and cell cycle.Results The mouse pcDNA3.0/p27kip1 eukaryotic expression plasmid were successfully constructed.The cells transfected with pcDNA3.0/p27kip1 expressed more p27 protein,and their proliferation was inhibited obviously.The percentage of the cells in G0/G1 phase increased,and that in S phase decreased significantly.Conclusions Mouse pcDNA3.0/p27kip1 eukaryotic expression plasmid could be expressed in mononuclear cells,and overexpression of p27 protein could inhibit mononuclear cell proliferation by restricting the cell cycle,making cell cycle arrest in G0/G1 phase.