可稳定表达ZsGreen1基因的正常细胞来源单核细胞株的构建

Establishment of Normal Cell-Derived Monocyte Cell Line Stably Expressing the ZsGreen1 Gene

为探索构建可表达外源基因的正常细胞来源单核细胞株的可行方案,该研究分别尝试以脂质体转染法、腺病毒载体感染法和含Tet-On调控元件的慢病毒载体感染法,构建可表达绿色荧光蛋白的SC细胞株。经慢病毒感染和靶向扩增获得稳定表达目的基因的SC细胞后,该研究进而验证了该细胞可否在PMA诱导下分化为典型的巨噬细胞。研究结果显示,脂质体转染法和腺病毒载体感染法未能有效导入外源基因至SC细胞;经慢病毒感染和靶向扩增,该研究成功构建了2株可稳定表达ZsGreen1基因的SC细胞株(SC-ZsGreen1),其ZsGreen1阳性表达率均高于95%; SCZsGreen1细胞与SC细胞经PMA处理后均具相似的巨噬细胞表型特征,包括CD11b和CD14表达升高、吞噬能力上调和趋化因子IL-8分泌水平上升。综上,该研究报道了一种构建可稳定表达外源基因的正常人外周血来源单核细胞株的可行方案。

It is demanded in the field of immunology to establish monocyte cell lines derived from normal human cells that stably express exogenous genes. To address this question, we employed multiple methods to transfect the ZsGreenl gene into the normal human SC monocytic cells, including Lipofectamine 3000, adenovirus and Tet-on element containing lentivirus. We found that the transfection/infection rates by using Lipofectamine 3000 and adenovirus in SC cells were 0.66% and 0%, respectively. The infection rate of Tet-on lentivirus was 8.5% in SC cells. ZsGreenl＋ SC cells （SC-ZsGreenl cells） were then screened out and cultured, resulting in the acquisition of two SC-ZsGreenl cell strains. Both stains reached over 95% of the positive ZsGreenl expression rate. Next, we used PMA to allow these cells to differentiate into macrophages. In addition to the upregulated phagocytotic effect, PMA introduced increased CD1 lb and CD 14 expression, and promoted IL-8 secretion in both SC-ZsGreenl cell stains, similar to SC cells. In conclusion, we reported a feasible strategy for the establishment of normal cell-derived monocyte cell lines stably expressing the exogenous gene of ZsGreenl.