摘要
目的检测NCSTN基因稳定沉默的人永生化角质形成细胞(HaCaT)增殖活性及相关分化蛋白的改变,初步探索反常性痤疮发病的可能机制。方法构建慢病毒介导shRNA沉默NCSTN基因的HaCaT细胞模型(shRNA组),转染空载慢病毒的HaCaT细胞作为阴性对照组,实时定量PCR和Western印迹法检测NCSTN基因的沉默效率。CCK8法检测HaCaT细胞增殖活性,实时定量PCR和Western印迹法检测HaCaT细胞角蛋白(CK1、CK5、CK7、CK10、CK14、CK16、CK17、CK18、CK19、CK20)及其他分化分子(内披蛋白、兜甲蛋白)mRNA和蛋白的表达。两组计量资料的比较采用两独立样本t检验。结果shRNA组NCSTN mRNA及蛋白表达(0.42±0.19、0.30±0.07)均显著低于阴性对照组(1.00±0.34、1.00±0.26;t=5.196、2.637,P<0.001、<0.05),基因沉默效率达70%。与阴性对照组相比,shRNA组HaCaT细胞明显增殖活跃,CK16、CK19蛋白表达显著下调(t=3.787、3.817,P<0.01、<0.05),终末分化分子内披蛋白表达明显降低(t=2.904,P<0.05)。结论稳定沉默NCSTN基因表达会引起HaCaT细胞异常增殖分化,为后续探究NCSTN基因突变引起反常性痤疮提供新思路。
Objective To evaluate the proliferative activity of and changes in the expression of related differentiation proteins in a stably NCSTN gene-silenced human immortalized keratinocyte cell line HaCaT,and to preliminarily explore the possible mechanism underlying the occurrence of acne inversa.Methods By lentivirus-mediated short hairpin RNA(shRNA),a NCSTN gene-silenced HaCaT cell model was established(shRNA group),and other HaCaT cells transfected with empty lentivirus served as a negative control group.Real-time quantitative PCR and Western blot analysis were performed to determine the NCSTN gene-silencing efficiency.Cell counting kit-8(CCK8)assay was conducted to evaluate the proliferative activity of HaCaT cells,and real-time quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of cytokeratins(CK1,CK5,CK7,CK10,CK14,CK16,CK17,CK18,CK19 and CK20)and other differentiation molecules(involucrin and loricrin)respectively in HaCaT cells.Two-independent-sample t test was used to compare the measurement data between two groups.Results NCSTN mRNA and protein expression were significantly lower in the shRNA group(0.42±0.19,0.30±0.07 respectively)than in the negative control group(1.00±0.34,1.00±0.26;t=5.196,2.637,P<0.001,<0.05,respectively),and the gene-silencing efficiency was 70%.Compared with the negative control group,the shRNA group showed higher cellular proliferative activity,but decreased protein expression of CK16,CK19 and terminal differentiation molecule involucrin(t=3.787,3.817,2.904,P<0.01,<0.05,<0.05,respectively).Conclusion Stable silencing of NCSTN gene can lead to abnormal proliferation and differentiation of HaCaT cells,which provides new ideas for subsequent exploration of acne inversa caused by NCSTN gene mutation.
作者
张婉璐
张媛媛
吴英达
程萍
李文锐
徐浩翔
王宝玺
何艳艳
李诚让
Zhang Wanlu;Zhang Yuanyuan;Wu Yingda;Cheng Ping;Li Wenrui;Xu Haoxiang;Wang Baoxi;He Yanyan;Li Chengrang(Department of Dermatology,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China;Department of Dermatology,Plastic Surgery Hospital,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100144,China;Department of Dermatology,The First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,Anhui,China)
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2020年第9期704-709,共6页
Chinese Journal of Dermatology
基金
国家自然科学基金(81472872)
中国医学科学院医学与健康科技创新工程项目(2016-I2M-1-002)
北京协和医学院中央高校基本科研业务费项目(3332019160)。
