Egr-1基因调控序列连接OSM cDNA表达质粒的构建及其在黑色素瘤细胞中的表达

ＯＳＭ是一种对黑色素瘤细胞显示抑制作用的细胞因子．为进行ＯＳＭ针对黑色素瘤的基因－放射治疗研究，构建了小鼠Ｅｇｒ－１基因调控序列引导入ＯＳＭｃＤＮＡ真核表达质粒（ｐＥＯ），ｐＥＯ质粒转染小鼠Ｂ－１６黑色素瘤细胞，经Ｇ４１８和抗人ＯＳＭ抗体的筛选，获得了稳定表达ＯＳＭ的克隆细胞（ｐＥＯ－１细胞），ＯＳＭ表达量可达５．９７ｎｇ每１０５细胞天，分子量为３２ｋＤ．ｐＥＯ－１细胞用一定浓度Ｈ２Ｏ２处理后ＯＳＭ表达量可提高６２％，表明ｐＥＯ重组质粒可在氧自由基的刺激作用下增强ＯＳＭ表达

Construction and Expression of the Human OSM cDNA Recombinant Vector Directed by Mouse Egr 1 Gene Promoter in Melanoma Cells

Oncostatin M(OSM),a single chain glycoprotein cytokine produced by stimulated macrophages and T lymphocytes,was previously identified by its ability to inhibit in vitro the proliferation of cells from melanoma and other solid tumors.To prove the potential application of OSM in the gene radiotheraphy for melanoma in vivo,a human OSM cDNA recombinant expression vector directed by Egr 1 gene promoter (pCl neo Egr 1R hOSM cDNA,pEO) was constructed and used to transfect mouse B 16 melanoma cells.The cloned cells secreting OSM continuously had been selected with G418 and anti hOSM antibodies,and the amount of secreted OSM in serum free supernatants was about 5 97 ng(per day 10 5 cells) determined by ELISA and with an apparent molecular weight of 32 kD by SDS electrophoresis and Western blot.It was also found that 62% of increased secretion of OSM from the pEO transfected cells was achieved as compared with control levels when H 2O 2 was added in medium,indicating that the Egr 1R hOSM construct could be activated by oxygen radicals so as to enhance the expression of OSM gene.