Effect of NCSTN gene silencing on the proliferation and differentiation of HaCaT cells / 中华皮肤科杂志

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.

Three cases of dermatomyositis presenting with inverse Gottron's papules / 中华皮肤科杂志

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.

Construction of a Nicastrin gene-silenced human immortalized keratinocyte cell model by lentivirus-mediated short hairpin RNA / 中华皮肤科杂志

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.

Pharmacoeconomics Evaluation of Azithromycin vs. Amoxicillin Clavulanate in the Treatment of Lower Respi-ratory Tract Infection / 中国药房

OBJECTIVE:To evaluate the economics of azithromycin vs. amocillin clavulante in the treatment of lower respirato-ry tract infections. METHODS:System evaluation was adopted to retrieve the randomized controlled trials(RCT)about azithromy-cin(test group)vs. amoxicillin clavulanate(control group)in the treatment of lower respiratory tract infections. Information was col-lected and Meta-analyses were performed. On this basis and short-term decision tree model,cost factors were added to conduct the pharmacoeconomics by the principle of PICO of Treeage Pro 2011 edition software. RESULTS:Totally 18 RCT were enrolled,in-volving 3 365 patients. Results of Meta-analysis showed that there were no significant differences in the effective rate [RR=0.93, 95%CI(0.55,1.55),P=0.77] and incidence of adverse reactions [RR=0.79,95%CI(0.62,1.0),P=0.05] between 2 groups. The av-erage treatment cost in test group and control group was respectively 790.4 yuan and 884.4 yuan,and cost-effectiveness ratio was respectively 216.0 and 245.7,and the incremental cost-effectiveness ratio(ICER)was -1 392.59. CONCLUSIONS:Azithromycin has similar efficacy and safety to amoxicillin clavulanate in the treatment of lower respiratory tract infection,however,azithromy-cin has better cost-effectiveness.