摘要
目的将腺病毒Ad5F35-GFP、Ad5-GFP及LipofectamineTM2000对4种细胞的转染效率进行比较,以获得Ad5F35-GFP适用的细胞种类及各类细胞适用的转染方法。方法采用携带绿色荧光蛋白(Green fluorescent protein,GFP)基因的Ad5F35-GFP、Ad5-GFP及LipofectamineTM2000,分别转染人慢性粒细胞白血病细胞K562、人外周血单个核细胞(PBMC)、小鼠脂肪细胞3T3-L1和肝癌细胞Hep A1-6,于转染48 h后,倒置荧光显微镜下观察转染效率,RT-PCR法检测GFP基因mRNA的转录水平。结果 Ad5F35-GFP转染K562细胞、PBMC效率均较高,分别为61%和56%,但不能有效地转染3T3-L1(5%)、HepA1-6(15%)细胞;Ad5F35-GFP、Ad5-GFP及LipofectamineTM2000均不能有效地转染3T3-L1细胞,转染效率分别为5%、5%和6%;Ad5-GFP、LipofectamineTM2000转染Hep A1-6细胞效率均较高,分别为73%和71%。结论 Ad5F35-GFP可有效转染人造血细胞和单核细胞,为相关基因的研究及治疗领域的应用奠定了基础。
Objective To comparing the transfection efficiencies adenoviruses Ad5-GFP,Ad5F35-GFP and LipofectamineTM 2000 to four cell lines so as to obtain the suitable cell line for Ad5F35-GFP and the suitable transfection method for various cells.Methods Human chronic granulocytic leukemia K562 cells,human peripherhal blood mononuclear cells（PBMCs）,mouse adipocyte 3T3-L1 strain and mouse liver cancer Hep A 1-6 cells were transfected with adenoviruses Ad5-GFP,Ad5F35-GFP and plasmid pEGFP-N1（with LipofectamineTM 2000）,separately.The transfection efficacy was observed under invert fluorescent microscope 48 h after transfection,while the transcription level of GFP mRNA was determined by RT-PCR.Results Ad5F35-GFP was transfected to K562 cells and PBMCs effectively,with transfection efficacies of 61% and 56% respectively,while was not effectively transfected to 3T3-L1（5%） and Hep A1-6（15%） cells.However,Ad5F35-GFP,Ad5-GFP and LipofectamineTM 2000 were not effectively transfected to 3T3-L1 cells,with transfection efficacies of 5%,5% and 6% respectively.The transfection efficacies of Ad5-GFP and Lipofectamine%TM 2000 to Hep A1-6 cells were 73% and 71% respectively.Conclusion Ad5F35-GFP may be effectively transfected to human hemopoietic cells and monocytes,which laid a foundation of study on relevant genes and their application in gene therapy.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第11期1444-1448,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金面上项目(81172016)
