稳定表达人GM-CSF和IL-4 EA.hy926细胞株建立及对DC诱导作用

CONSTRUCTION OF TWO EA.hy926 CELL LINES TO EXPRESS GM-CSF AND IL-4 AND THEIR INDUCTION EFFECT ON DC

目的获得分别能稳定表达人白细胞介素4(IL-4)和人粒细胞-巨噬细胞集落刺激因子(GM-CSF)的内皮细胞株,并用于树突状细胞(DC)的体外诱导培养。方法构建重组的慢病毒表达载体,在293T细胞中进行慢病毒的包装,用获得的高滴度的慢病毒感染内皮细胞EA.hy926细胞株,建立能够高效、稳定表达IL-4和GM-CSF的EA.hy926细胞株。结果 EA.hy926细胞株感染效率达90%以上,长期传代仍能保持较高阳性率。RT-PCR在mRNA水平上检测目的基因有表达;ELISA和Western方法证实感染后细胞IL-4、GM-CSF在蛋白水平的表达分别是未感染细胞的80倍和143倍。感染后细胞的生长状态良好,能够长期在体外进行传代培养,可以用于DC诱导实验。结论构建的细胞系可以稳定表达细胞因子IL-4和GM-CSF。

Objective To get endothelial cell line that stably express IL-4 and GM-CSF that can be used for inducing den- dritic cell （DC） culture in vitro. Methods A recombinant lentiviral expression factor was created, the lentivirus enclosure was carried out in 293T cells,the acquired high titer of the virus was used to infect EA. hy926 cells and create an EA. hy926 cell line that can effectively and stably express GM-CSF and IL-4. Results The efficiency of infection of EA. hy926 cell line was above 90%, a highly positive rate was still remained after a long-term subculture. The cells＇ mRNA was extracted by Trizol then processed RT-PCR to detect the expression of the two target genes; the expression of IL-4 and GM-CSF at the protein level was 80 and 143 fold by ELISA analysis. The two infected cell lines had a good growth state, and serial suhcultivation could be conducted, which could be used for inducing experiment of DC. Conclusion The two infected EA. hy926 cell lines can stably express GM-CSF and IL-4.