RI基因真核表达载体的构建及其对人脐静脉内皮细胞的影响.docVIP

RI基因真核表达载体的构建及其对人脐静脉内皮细胞的影响 李红彦，潘湘阳，姚 雪，熊东梅, 陈俊霞 （400016 重庆，重庆医科大学细胞生物学与遗传学教研室，分子医学与肿瘤研究中心) [摘要] 目的 构建融合表达载体pcDNA3.1–RI，并检测核糖核酸酶抑制因子(ribonuclease inhibitor，RI)基因与血管生成素(angiogenin, ANG)的关系及对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVEC)增殖及迁移能的影响。 方法 用RT-PCR方法扩增了RI基因，酶切后将其插入pcDNA3.1–，构建融合表达载体pcDNA3.1–RI，在脂质体介导下转染人脐静脉内皮细胞HUVEC，通过RT-PCR检测RI、ANG基因的mRNA表达水平，Western blot检测RI、ANG及影响内皮细胞迁移能力重要的2个蛋白MMP-2、MMP-9的表达水平，CO-IP法检测ANG和RI的相互作用，MTT法检测细胞的增殖活力，流式细胞术检测细胞周期的分布。 结果 真核表达质粒构建成功；实验组pcDNA3.1-RI细胞RI基因的mRNA及蛋白的表达较2个对照组(转pcDNA3.1–空和未转染质粒组)比较,均呈显著性增加(P0.05)，转染RI基因的细胞ANG基因的mRNA及蛋白的表达均降低(P0.05)；MMP-2、MMP-9基因的蛋白表达水平降低(P0.05)；CO-IP法检测到ANG和RI在细胞内能结合；转染pcDNA3.1–RI质粒到HUVEC细胞后细胞的增殖活力明显降低(P0.05),G0～G1期比例明显增加，S期减少。 结论 成功构建的真核表达质粒能显著增加RI基因及其蛋白水平的表达，RI可以直接在转录水平上降低ANG的表达，在细胞内与ANG结合，从而影响内皮细胞的增殖、迁移能力。 [关键词] 核糖核酸酶抑制因子；人脐静脉内皮细胞；血管生成素；真核表达 [中图法分类号] [文献标志码] A Construction of eukaryotic expressive vector of RI gene and the effects of the transfected pcDNA3.1-RI on HUVEC cells Li Hongyan , Pan Xiangyang,Yao Xue, Xiong Dongmei, Chen Junxia（Cell Biology and Genetics Division,Molecule Medical and Tumor Research Center,Chongqing Medical University,Chongqing, 400016，China） [Abstract] Objective To clone and construct an eukaryotic expressive vector of ribonuclease inhibitor(RI)gene, as well as to observe the effects of the transfected PCdna3.1-RI on the growth of HUVEC cells and the relation between RI and ANG. Methods A segment of RI gene augmented by RT-PCR was digested by special enzyme and cloned into pcDNA3.1–. The vectors of pcDNA3.1–RI and pcDNA3.1– were transfected into HUVEC cells respectively. The mRNA and protein expression level of the RI, ANG,MMP-2 and MMP-9 gene were tested by RT-polymerase chain reaction (PCR), Western blotting.The relationship of ANG mRNA was detected by CO-IP method. HUVEC cells proliferation ability were detected by MTT and cell flow cytometry. Results Enzyme digestion and DNA sequencing verified the constructed vectors were correct.