阳离子脂质体介导hGM-CSF真核表达载体在骨髓基质细胞系中的表达

Expression of lipofect AMINE mediated human GM-CSF eukaryotic expressing vector in HFCL cells

目的 构建人粒 巨噬细胞集落刺激因子 (hGM CSF)真核表达载体pIRES1neo hGM CSF ,并观察其在人骨髓基质细胞系HFCL中的表达。方法 用DNA重组技术 ,将hGM CSFcDNA插入到真核表达载体pIRES1neo的多克隆位点中 ,获得阳性重组载体pIRES1neo hGM CSF。以脂质体介导法转染人骨髓基质细胞系HFCL细胞 ,筛选获得的阳性细胞用Southernblot和Northernblot方法分别检测其基因组DNA整合和mRNA转录 ,用ELISA及依赖株 (TF 1)法检测其蛋白表达量和活性。结果 酶切鉴定表明成功地构建了重组真核表达载体pIRES1neo hGM CSF ;hGM CSF基因已整合到HFCL细胞基因组DNA中 ,在mRNA和蛋白质水平上均可检测到其表达 ;hGM CSF的表达量为 (5 6 .9± 0 .7)ng 10 6细胞 ,分泌量在细胞传代 30次后仍保持稳定 ,活性为 (6 .5 6± 0 .16 )× 10 3 U 10 6 细胞。结论 所构建的hGM CSF真核表达载体在人骨髓基质细胞系HFCL细胞中获得持续稳定表达 ,并具有良好的生物学活性 ,为细胞因子基因治疗的体内实验研究提供了实验依据。

Objective To construct an eukaryotic expressing vector pIRES1neo/hGM CSF and express it in human bone marrow stromal cell line HFCL. Methods Human granulocyte/macrophage colony stimulating factor cDNA ( hGM CSF cDNA,751?bp) was inserted into an effective eukaryotic expressing vector pIRES1neo which contains the human cytomegalovirus (CMV) major immediate early promoter/enhancer and the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV). HFCL cells were transfected with the recombinant vector pIRES1neo/hGM CSF by liposome mediated gene transfer method.Integration of hGM CSF in the genome,transcription of its mRNA and expression of its protein in the transfected HFCL cells were assayed by Southern blot, Northern blot, ELISA and hGM CSF dependent cell line TF 1. Results hGM CSF cDNA was integrated into HFCL genome successfully,hGM CSF mRNA was transcripted and hGM CSF protein was expressed of (56.9±0.7) ng/10 6 cells by ELISA and (6.56±0 16)×10 3 U/10 6 cells per day by TF 1 cell assay in the supernatant. Conclusion The recombinant vector is proved to be stably expressed in HFCL cells and the biological activity of hGM CSF was detectable in the supernatant of the transfected cells.