摘要
目的在人骨髓间充质干细胞内大量稳定的表达人白血病抑制因子(human Leukemia Inhibitory Factor,hLIF)。方法应用RT-PCR方法从人蜕膜组织中扩增出白血病抑制因子,并将其构建于pcDNA3.1真核表达载体上。利用脂质体进行基因骨髓间充质干细胞的转染。利用G418进行基因的稳定表达筛选。通过RT-PCR和Western blot方法进行基因表达的检测。结果成功扩增出人白血病抑制因子基因,成功构建hLIF-pcDNA3.1真核表达载体。转染LIF-pcDNA3.1的骨髓间充质干细胞经G418筛选,存活十代以上。经RT-PCR及Western blot方法检测发现转染LIF-pcD-NA3.1的骨髓间充质干细胞表达白血病抑制因子明显高于未转染的骨髓间充质干细胞。结论成功构建LIF-pcD-NA3.1的真核表达载体并使其在骨髓间充质干细胞得到稳定表达。
Objective To contruet eukaryotic expression vector pcDNA3.1-L IF and express stablely LIF in the bone marrow derived mesenehymal stem cells.Methods LIF gene was cloned from pregnant uterine decidua tissues by using RT-PCR method and was inserted into pcDNA3.1 to construct eukaryotie expression vector consisting of LIF gene. then transfeeted by lipofectamine into BMSCs. LIF expression was analyzed using Western blot and RT-PCR. Results The recombinant vector pcDNA3.1-L IF was identified by restriction enzyme analysis and PCR amplification. LIF was suceessfully expressed in BMSCs. Conclusion The eukaryotie expression vector pcDNA3.1-L IF, which can be expressed stablely in BMSCs, has been successfully constructed.
出处
《中国实验诊断学》
北大核心
2009年第1期15-18,共4页
Chinese Journal of Laboratory Diagnosis
