小鼠干细胞因子基因以脂质体介导转染脐血细胞

Study on Lipofectin^(RM) mediated murine SCF gene transduction into umbilical blood cells

目的小鼠干细胞因子 (SCF)以脂质体介导转染脐血细胞并表达。方法采用PCR技术从 pRC/CMV(含SCFcDNA)中钓取SCFcDNA膜外段活性区 ,克隆入真核表达载体 pcDNA3 ,转染传 2代的脐血细胞 ;用RT PCR方法检测mRNA表达水平及通过协同刺激集落形成来检测转染细胞上清的生物活性。结果转染SCFcDNA的脐血细胞SCFmRNA水平表达量高于对照组 ,其细胞培养上清协同GM CSF刺激骨髓细胞形成集落数比GM CSF单独作用高两倍左右。结论脂质体介导质粒载体转染培养富集的脐血细胞并表达 ,且转染上清具有生物学活性 ,为进一步研究转染基因的表达特性及持续时间提供依据。

PurposeThe aim is to study the transduction of murine stem cell factor(SCF) into umbilical blood cells by Lipofectin RM mediated and expression in them.MethodsMurine SCF cDNA encoding extra cellular domain was isolated using PCR from plasmid pRC/CMV(containing SCF gene), and then recombined into the expression vector pcDNA3,and transferred into enrichment cultural umbilical blood cells. Semi quantitative RT PCR was used to examine the expression on mRNA level.And the effects of supernatant of transfected umbilical blood cells were investigated alone or in coordinate with GM CSF by colony formation test of human bone marrow cells in semisolid culture.ResultsSCF mRNA was expressed in transfected umbilical blood cells.The supernatant of transfected umbilical blood cells could increase the number of CFU GM, synergizing with GM CSF.ConclusionThe supernatant of umbilical blood cells transfected with vector containing mSCF gene can stimulate the colony formation of human bone marrow cells in combination with GM CSF.