早老蛋白增强子2基因沉默对HaCaT细胞增殖及γ分泌酶表达的影响  

Effect of PSENEN gene silencing on the proliferation of andγ-secretase expression in HaCaT cells

作　　者：李文锐 张媛媛 贾苇雪 何艳艳 徐浩翔 林麟 李诚让 Li Wenrui;Zhang Yuanyuan;Jia Weixue;He Yanyan;Xu Haoxiang;Lin Lin;Li Chengrang(Department of Dermatology,Hospital of Dermatology,Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing 210042,China;Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,Nanjing 210042,China)

机构地区：[1]中国医学科学院、北京协和医学院皮肤病医院皮肤科,南京210042 [2]江苏省皮肤病与性病分子生物学重点实验室,南京210042

出　　处：《中华皮肤科杂志》2021年第4期318-324,共7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)。

摘　　要：目的构建早老蛋白增强子2(PSENEN)基因沉默的人永生化角质形成细胞(HaCaT)模型,探究PSENEN基因表达下调对HaCaT细胞增殖以及γ分泌酶表达的影响。方法设计3组靶向PSENEN基因的shRNA序列,与线性化的LV3-pGLV-h1-GFP-puro载体构建慢病毒重组表达质粒,经酶切及测序鉴定后,进行慢病毒的包装、纯化和滴度测定。将HaCaT细胞分为5组进行转染:shRNA1组、shRNA2组、shRNA3组分别加入含沉默PSENEN基因的shRNA1、shRNA2、shRNA3慢病毒原液,NC组加入含阴性对照shNC慢病毒原液,空白组不加病毒液。转染完成后,荧光显微镜下观察荧光表达,流式细胞仪检测转染效率。CCK8法测定HaCaT细胞增殖活性,实时荧光定量PCR(qPCR)、Western印迹法分别检测PSENEN、呆蛋白(NCT)、早老蛋白1(PS1)、前咽缺陷蛋白1a(APH1a)mRNA、蛋白的相对表达量。统计分析采用重复测量方差分析、单因素方差分析,组间两两比较采用LSD-t检验。结果倒置荧光显微镜下观察到shRNA1~shRNA3组、NC组均有荧光表达,流式细胞仪示转染效率均达98%以上。qPCR、Western印迹法显示,与NC组(1.054±0.272、1.076±0.075)相比,shRNA1、shRNA2、shRNA3组PSENEN mRNA(0.187±0.010、0.163±0.022、0.174±0.009)、蛋白(0.219±0.097、0.208±0.014、0.185±0.062)表达均显著降低(均P<0.001)。CCK8法显示,与NC组相比,shRNA1组细胞增殖活性在0、12、36、48 h均显著增高(均P<0.05),24和60 h差异无统计学意义(均P>0.05);shRNA2、shRNA3组在0、12、24、36、48、60 h细胞增殖水平均显著高于NC组(均P<0.05)。shRNA1组、shRNA2组、shRNA3组、NC组、空白组间NCT、PS1、APH1a基因mRNA表达差异无统计学意义(F值分别为8.168、4.644、1.981,均P>0.05),mNCT、imNCT、PS1-CTF、APH1a蛋白相对表达差异有统计学意义(F值分别为39.268、5.929、27.842、20.663,均P≤0.01)。与NC组相比,shRNA1、shRNA2、shRNA3组mNCT、PS1-CTF、APH1a蛋白表达均显著降低(均P<0.01),imNCT蛋白表达变�Objective To establish a presenilin enhancer-2(PSENEN)gene-silenced human immortalized keratinocyte(HaCaT)cell model,and to evaluate the effect of PSENEN gene silencing on the proliferation of andγ-secretase expression in HaCaT cells.Methods Three shRNAs targeting the PSENEN gene were constructed,and inserted into the linearized LV3-pGLV-h1-GFP-puro vector to establish a recombinant lentiviral expression plasmid.After restriction enzyme digestion and sequencing,lentiviral packaging and purification were performed,and lentiviral titer was determined.Cultured HaCaT cells were divided into 5 groups:shRNA1,shRNA2 and shRNA3 groups treated with the lentivirus solutions containing PSENEN gene-targeted shRNA1,shRNA2 and shRNA3 respectively,NC group treated with the lentivirus solution containing a negative control shRNA(shNC),and blank group treated without lentivirus solution.After transfection,inverted fluorescence microscopy was performed,and transfection efficiency was determined by flow cytometry.Cell counting kit-8(CCK8)assay was performed to evaluate the effect of PSENEN gene silencing on the proliferation of HaCaT cells,and real-time fluorescence-based quantitative PCR(qPCR)and Western blot analysis were conducted to determine the mRNA and protein expression of PSENEN,nicastrin(NCT),presenilin-1(PS1)and anterior pharynx defective 1a(APH1a)genes respectively.Statistical analysis was carried out by using repeated measures analysis of variance,one-way analysis of variance,and least significant difference t test for multiple comparisons.Results Inverted fluorescence microscopy showed that fluorescence was observed in the shRNA1 group,shRNA2 group,shRNA3 group and NC group,and flow cytometry showed that the transfection efficiency was over 98%in the above 4 groups.qPCR and Western blot analysis revealed that the mRNA and protein expression of PSENEN gene significantly decreased in the shRNA1(0.187±0.010,0.219±0.097,respectively),shRNA2(0.163±0.022,0.208±0.014,respectively)and shRNA3(0.174±0.009,0.185±0.062,res

关 键 词：化脓性汗腺炎 基因沉默 细胞增殖 PSENEN基因 Γ分泌酶 HaCaT细胞 

分 类 号：R758.733[医药卫生—皮肤病学与性病学]