应用绿色荧光研究人抑瘤素基因在CHO细胞中的表达  

Employing green fluorescence to investigate expression of human Oncostatin M in CHO cell line

作　　者：李晓利[1] 王强[2] 许红民[1] 赵彦林[3] 赵士富[3] 尉承泽[4] 

机构地区：[1]解放军总医院第309临床部,北京100091 [2]解放军总医院,北京100853 [3]军事医学科学院二所十室,北京100850 [4]军事医学科学院附属307医院,北京100039

出　　处：《军医进修学院学报》2005年第6期404-406,共3页Academic Journal of Pla Postgraduate Medical School

摘　　要：目的:建立一种简易、快速检测细胞内外源性基因稳定表达的方法。方法:以携带绿色荧光蛋白的真核表达质粒pEGFP-N1为骨架,将完整的hOSM基因ORF克隆到pEGFP-N1上游的多克隆位点,构建表达载体pOSM-EGFP-N1,脂质体介导转染中国仓鼠卵巢细胞(CHO),G418筛选,挑选阳性克隆。荧光显微镜、流式细胞术分析转染了pOSM-EGFP-N1质粒的CHO细胞纯度。RT-PCR的方法分析OSM基因水平表达。结果:成功建立稳定表达OSM的CHO细胞株(CHO-OSM-EGFP),纯度达到99.76%,且OSM基因在CHO-OSM-EGFP细胞中稳定表达。结论:构建了携带绿色荧光、表达OSM的pOSM-EGFP-N1质粒转染的CHO细胞,只通过荧光显微镜观察和FCM检测细胞绿色荧光,即可明确OSM转染成功。为表达外源性基因阳性细胞的筛选提供了简易的方法。Objective： To establish a simple and rapid method for measuring the exogenous gene expression in eukaryotic cells. Methods. The plasmid that expresses green fluorescence, pOSM-EGFP-N1 was constructed by the insertion of the complete ORF of human oncostatin M （OSM） into pEGFP-N1 vector, and transfect Chinese hamster ovary （CHO） cells to get CHO-OSM-EGFP cells. After screening with G418 culture, the colony of expression green fluorescence was picked up under the fluorescence microscope. The CHO-OSM-EGFP cell line was obtained by continuously cultivation. Purity of cell line was checked by flow cytometry（FCM）. The OSM mRNA expression was examined by RT-PCR. Results： The CHO-OSM-EGFP cell line is stable expresses green fluorescence and the cell colonies with uniformly fluorescent intensity in culture and easily observed by fluorescence microscope. The purity of the CHO-OSM-EGFP cell line is 99.76% that analysis by FCM. The target gene OSM expression was detected in CHO-OSM-EGFP cell line by RT-PCR. Conclusion： These results showed that inserting the OSM gene into an expression green fluorescence vector, pEGFP-N1. The plasmid pOSM-EGFP-N1 transfect CHO cells, we can get stable expresses green fluorescence CHO-OSM-EGFP cell line. Through the green fluorescence expression, we will identify the exogenous gene expression in the eukaryotic cells.

关 键 词：抑瘤素 荧光 基因表达 CHO细胞 

分 类 号：R338[医药卫生—人体生理学]