人髓核细胞中不同滴度慢病毒介导绿色荧光蛋白基因的表达

Green fluorescence protein gene expression in human nucleus pulposus cells mediated by different titers of lentivirus

背景:慢病毒作为基因载体感染椎间盘髓核细胞的感染效率研究具有实际应用价值。目的:测定不同滴度慢病毒介导的绿色荧光蛋白基因感染人髓核细胞的感染条件及感染参数。方法:采用酶消化法分离培养人髓核细胞,利用基因重组技术构建高滴度慢病毒介导的绿色荧光蛋白基因,按照不同感染复数感染第2代人髓核细胞。结果与结论:第2代髓核细胞在慢病毒介导的绿色荧光蛋白基因的感染复数为1,10,50,100时,感染后第4天荧光显微镜观察,流式细胞术测定其感染效率分别为32.1%,41.1%,54.2%和86.8%,继续传代培养3代,第5代椎间盘髓核细胞绿色荧光蛋白的表达仍能保持在60%以上。表明慢病毒介导的绿色荧光蛋白基因在人髓核细胞中可以持续高效表达。

BACKGROUND：In the study of intervertebral disc tissue engineering,the intervertebral disc cells modified by gene engineering have the advantages of fast growth and secreting more extracellular matrix as the seed cells.Investigating the infection efficiency of intervertebral disc nucleus pulposus（NP） cells infected by lentivirus will have practical value.OBJECTIVE：To detect the infection condition and infection parameter of human NP cells infected by different titers of green fluorescence protein mediated by lentivirus.To provide the experimental evidence for the lentivirus infection as gene vector to the human NP cells.METHODS：Human NP cells were isolated and cultured from two idiopathic scoliosis patients via enzyme digestion.High titer of lentivirus-mediated green fluorescence protein（GFP） gene was constructed by gene reconstitution technique.The passage 2 NP cells were infected with different multiplicities of infection（MOIs）,and green fluorescence was observed using an inverted fluorescence microscope.The infection efficiency was detected by flow cytometry.RESULTS AND CONCLUSION：After 4 days,the infection efficiency of the passage 2 NP cells infected with different MOIs（1,10,50 and 100） was 32.1%,41.1%,54.2% and 86.8%,respectively.The expression of GFP in the NP cells was maintained above 60% until the passage 5.The GFP gene mediated by lentivirus was effectively expressed in human NP cells for a long time period.It may provide evidence that lentivirus-mediated GFP gene is highly effectively expressed in human NP cells.