关键词: 模型, 细胞

Abstract: Mao Qiuxia, Zhang Wanlu, He Yanyan, Jia Weixue, Yang Brooks, Li Li, Li Liming, Zhang Xiaofeng, Xu Haoxiang, Chen Xu, Wang Baoxi, Li Chengrang Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Nanjing 210042, China （Mao QX ［current affiliation: Department of Dermatology, Jiangyin Hospital of Traditional Chinese Medicine, Affiliated to Nanjing University of Chinese Medicine, Jiangyin 214400, Jiangsu, China］, Zhang WL, He YY, Li L, Li LM, Zhang XF, Xu HX, Chen X, Li CR）; Department of Dermatology, Henan Provincial People′s Hospital, Zhengzhou 450003, China （Jia WX）; Cutaneous Biology Research Center, Massachusetts General Hospital （Yang Brooks）; Department of Dermatology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100144, China （Wang BX） Corresponding author: Li Chengrang, Email: nylcr72@163.com 【Abstract】 Objective To construct a lentiviral vector delivering the Nicastrin （NCT） gene?targeted short hairpin RNA （shRNA） and determine gene?silencing efficiency of the vector in the human immortalized keratinocyte cell line HaCaT, and to construct a NCT gene?silenced HaCaT cell model to lay an experimental foundation for subsequently studying effects of NCT gene silencing on biological behavior of keratinocytes. Methods Three NCT gene?targeted shRNAs were designed and inserted into the pGLV3/H1/GFP + Puro vector to construct three recombinant plasmids, which were then confirmed by sequencing. Recombinant plasmids combined with lentivirus packaging plasmids were co?transfected into 293T cells to obtain lentivirus particles, and the virus titer was determined. Cultured HaCaT cells were divided into 3 groups: blank group receiving no treatment, negative control group infected with the empty vector LV3?shNC, interference groups infected with lentivirus NCT?shRNA1, ?shRNA2, ?shRNA3, respectively. Flow cytometry was performed to determine transfection efficiency, and real?time fluorescence?based quantitative PCR （qRT?PCR） and Western blot analysis were conducted to determine efficiency of target gene silencing in HaCaT cells, so as to select the most efficient interference sequence. Results Sequencing analysis indicated that recombinant lentiviral vector NCT?shRNA was constructed successfully. After co?transfection of recombinant plasmids and lentivirus packaging plasmids into 293T cells, the titer of recombinant lentivirus particles was about 109 TU/ml. Flow cytometry showed that the transfection efficiency was greater than 95%. qRT?PCR revealed that the NCT mRNA was obviously down?regulated in the interference group compared with the negative control group, and NCT?shRNA1 was the most efficient sequence with interference efficiency being 75%. Western blot analysis showed that the inhibition rate of NCT protein was 71.7% in the shRNA1 group compared with the negative control group. Conclusion The most efficient NCT?shRNA interference sequence is screened out, and the recombinant lentiviral vector NCT?shRNA and an NCT gene?silenced HaCaT cell model are both constructed successfully.

Key words: Models, cell