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

发布时间：2018-04-30 03:01

本文选题：核糖核酸酶抑制因子 + 人脐静脉内皮细胞　；参考：《重庆医科大学》2012年硕士论文

【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 endothelialcells, 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 空载体组和未转染质粒组)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。
[Abstract]:Objective to construct the fusion expression vector pcDNA3.1 ri and to detect the relationship between ribonuclease inhibitor ribonuclease receptor and angiogenin (Ang) and its effect on proliferation and migration of human umbilical vein endothelial cells (HUVECs). Methods RI gene was amplified by RT-PCR. The fusion expression vector pcDNA3.1 was constructed and transfected into human umbilical vein endothelial cells (HUVECs) mediated by liposome. The level of mRNA expression of RIANG gene was detected by RT-PCR. It is important to detect RIANG by Western blot and to affect the migration ability of HUVECs. The expression level of MMP-2MMP 9 was detected by CO-IP method. The interaction between ANG and RI was detected by MTT assay and the proliferative activity of cells was detected by MTT assay. The distribution of cell cycle was detected by flow cytometry. Results the expression of mRNA and protein of RI gene in pcDNA3.1-RI cells in the experimental group was compared with that in the control group (transfer pcDNA3.1 empty vector group and untransfected plasmid group), and the expression of RI gene in the experimental group was compared with that in the control group. The expression of mRNA and protein of ANG gene in the cells transfected with RI gene decreased, and the protein expression level of MMP-2MMP-9 gene decreased. The binding of ANG and RI in cells was detected by P0.05 ANG CO-IP method. After transfection of pcDNA3.1 / RI plasmid into HUVEC cells, the proliferative activity of the cells decreased significantly. The ratio of G _ 0 / G _ (1) phase of P0.05G _ (0) / G _ (1) increased significantly. Conclusion the successfully constructed eukaryotic expression plasmid can significantly increase the expression of RI gene and its protein. RI can directly reduce the expression of ANG at the transcriptional level and bind to ANG in cells, thus affecting the proliferation and migration ability of endothelial cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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