眼科新进展2018，Vol.38Issue(1)：27-30,4.DOI:10.13389/j.cnki.rao.2018.0005
丙酮酸激酶M1(PKM1)对眼黑色素瘤细胞迁移的影响
Effects of PKM1 gene on the migration of ocular melanoma cells
摘要
Abstract
Objective To construct the eukaryotic expression vector of pyruvate kinase M1 (PKM1) gene labeled with pXJ-40-myc and detect its biological activity in ocular B16 melanoma cells.Methods Ocular B16 melanoma ceils were randomly divided into experimental and control group,and the experimental group was transfected with pXJ-40-myc-PKM1 plasmid and the control group was transfected with pXJ-40-myc plasmid.Then PKM1 gene was amplified by PCR with human liver cDNA library as the template.The recombinant plasmid pXJ-40-myc-PKM1 was identified by bacteria PCR and double enzyme digestion,followed by transfection of pXJ40-myc-PKM1 and pXJ-40-myc plasmid into B16 melanoma cells,and finally,the expression of PKM1 protein was verified by the Western blot,while wound healing assay was used to detect the effects of PKM1 on the migration of ocular melanoma ceils.Results The length of PKM1 gene was 1800bp,which was consistent with the expected size.Compared with the control group,the result of bacteria PCR was positive.The length of double enzyme digestion was 4000 bp and 1800 bp respectively.Western blot results showed that recombinant plasmld pXJ-40-myc-PKM1 was successfully expressed in ocular B16 melanoma cells.Compared with the control group,wound healing assay showed that recombinant plasmid could inhibit the migration of ocular B16 melanoma cells.Conclusion The eukaryotic expression vector of pXJ-40-myc-PKM1 is successfully constructed,which can suppress the migration of ocular B16 melanoma cells.关键词
眼B16黑色素瘤细胞/丙酮酸激酶M1基因/克隆/真核表达Key words
ocular B16 melanoma cells/pyruvate kinase M1 gene/cloning/eukaryotic expression分类
医药卫生引用本文复制引用
刘园园,朱晓雨,刘莹,徐小洁,李玲,叶棋浓,刘丹..丙酮酸激酶M1(PKM1)对眼黑色素瘤细胞迁移的影响[J].眼科新进展,2018,38(1):27-30,4.基金项目
国家自然科学基金资助(编号:81472589、81672602、81402345、81502264).National Natural Science Foundation of China (No:81472589,81672602,81402345,81502264) （编号:81472589、81672602、81402345、81502264）
