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目的利用包装制备的重组慢病毒载体感染人脐带间充质干细胞,为后续的细胞重编程奠定基础。方法利用组织块贴壁法分离、培养、扩增出人脐带间充质干细胞(HUMSC)。携带目的基因(oct4、sox2、klf4、c-myc)及绿色荧光蛋白的质粒经验证、扩增后,转染HEK-293T细胞,制备携带上述基因的慢病毒上清液。荧光显微镜下梯度稀释法检测病毒滴度。慢病毒液感染3~5代生长良好的HUMSC,荧光显微镜及流式细胞术检测病毒感染力,RT-PCR及定量PCR检测目的基因mRNA水平。结果成功验证、扩增携带目的基因的质粒。制备的新鲜病毒液滴度达5×108U/mL,冻存后降为5×106U/ml。重组慢病毒感染HUMSC的感染力高于80%,4种目的基因的表达均明显高于对照。结论重组慢病毒载体可以在体外高效的转染HUMSC,并稳定表达目的基因,是HUMSC重编程的有效基因转移载体。
Objective To infect human umbilical cord mesenchymal stem cells with recombinant lentiviral vector prepared by packaging, which laid the foundation for subsequent cell reprogramming. Methods Human umbilical cord mesenchymal stem cells (HUMSC) were isolated, cultured and amplified by tissue block method. The plasmids carrying the target genes (oct4, sox2, klf4, c-myc) and green fluorescent protein were verified, amplified and transfected into HEK-293T cells to prepare lentivirus supernatants carrying these genes. Gradient dilution method was used to detect virus titer under fluorescence microscope. The lentiviral fluids were infected for 3 to 5 generations with well-developed HUMSC. The viral infectivity was detected by fluorescence microscopy and flow cytometry. The mRNA levels of the target genes were detected by RT-PCR and quantitative PCR. Results The plasmid containing the gene of interest was successfully verified and amplified. The prepared fresh virus had a titer of 5 × 108U / mL, which was reduced to 5 × 106U / ml after cryopreservation. The infectivity of recombinant lentivirus infected HUMSC was higher than 80%, and the expression of four kinds of target genes were significantly higher than that of the control. Conclusion The recombinant lentiviral vector can efficiently transfect HUMSC in vitro and stably express the target gene. It is a useful gene transfer vector for HUMSC reprogramming.