RESUMO
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 expression 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 expression 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.
RESUMO
Three cases of dermatomyositis presenting with inverse Gottron's papules were reported.Of the 3 patients,there were 2 males and 1 female aged 43,41 and 46 years respectively,whose disease durations were 1,6,7 months respectively.Inverse Gottron's papules manifested as papules overlying the palmar aspect of the metacarpal and interphalangeal joints,which were arranged in a linear pattern in 2 cases.Of the 3 cases,1 presented with interstitial pneumonia,3 with myositis,and 2 showed negative anti-nuclear antibody test.Histopathological examination of inverse Gottron's papules in 1 case revealed focal liquefaction degeneration of basal cells and perivascular infiltration with inflammatory cells,especially lymphocytes,in the superficial dermis.The 3 patients received oral glucocorticoids and immunosuppressive agents in the early period of treatment.After 3-month treatment,clinical symptoms including muscle weakness and muscle pain were improved evidently,but no obvious improvement in inverse Gottron's papules was observed.After 1-year treatment,these papules on the palmar aspect were markedly relieved in 1 case.