Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is sti...Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is still unknown,the current treatment is far from satisfactory.Thus,there is an urgent need to find a more effective therapy for this disease.Keratin 17(K17),a type I intermediate filament,is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis.Therefore,we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis.This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA(siRNA)on mice with imiquimod(IMQ)-induced psoriasis-like dermatitis.Methods:Eight-week-old female BALB/c mice were administered a 5%IMQ cream on both ears to produce psoriatic dermatitis.On day 3,K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days.The right ears of the mice were treated in parallel with negative control(NC)siRNA.Inflammation was evaluated by gross ear thickness,histopathology,the infiltration of inflammatory cells(CD3+T cells and neutrophils)using immunofluorescence,and the expression of cytokine production using real-time quantitative polymerase chain reaction.The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance.Results:The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears,as evidenced by the alleviated ear inflammation phenotype,including decreased ear thickness,infiltration of inflammatory cells(CD3+T cells and neutrophils),and inflammatory cytokine/chemokine expression levels(interleukin 17[IL-17],IL-22,IL-23,C-X-C motif chemokine ligand 1,and C-C motif chemokine ligand 20)(P<0.05 vs.the Blank or NC siRNA groups).Compared to the NC siRNA treatment,the K17 siRNA treatment resulted in increased K1 and K10 expression,which are characteristic of keratinocyte differentiation(vs.NC siRNA,K17 siRNA1 group:K1,t=4.782,P=0.0050;K10,t=3.365,P=0.0120;K17 siRNA2 group:K1,t=4.104,P=0.0093;K10,t=4.168,P=0.0042;siRNA Mix group:K1,t=3.065,P=0.0221;K10,t=10.83,P<0.0001),and decreased K16 expression,which is characteristic of keratinocyte proliferation(vs.NC siRNA,K17 siRNA1 group:t=4.156,P=0.0043;K17 siRNA2 group:t=2.834,P=0.0253;siRNA Mix group:t=2.734,P=0.0250).Conclusions:Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis.Thus,gene therapy targeting K17 may be a potential treatment approach for psoriasis.展开更多
目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成...目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。展开更多
目的观察白细胞介素-17A(IL-17A)对体外培养的人永生化角质形成细胞(Ha Ca T)角蛋白17(K17)表达及信号转导和转录激活因子3(STAT3)信号通路的影响。方法采用RPMI1640培养液培养Ha Ca T细胞,将细胞随机分为空白对照组、诱导组、抑制剂组...目的观察白细胞介素-17A(IL-17A)对体外培养的人永生化角质形成细胞(Ha Ca T)角蛋白17(K17)表达及信号转导和转录激活因子3(STAT3)信号通路的影响。方法采用RPMI1640培养液培养Ha Ca T细胞,将细胞随机分为空白对照组、诱导组、抑制剂组,空白对照组仅加DMEM高糖培养基,诱导组加入含50μg/L IL-17A的DMEM高糖培养基,抑制剂组加入含50μg/L IL-17A的DMEM高糖培养基和10μmol/L STAT3抑制剂Piceatannol。收集培养的Ha Ca T细胞,采用MTT比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,RT-PCR法检测细胞K17 mRNA表达水平,Western blotting法检测细胞K17和磷酸化STAT3(p-STAT3)蛋白表达水平。结果诱导组细胞增殖A值高于空白对照组和抑制剂组,细胞凋亡率低于空白对照组和抑制剂组(P均<0.01);诱导组K17mRNA及K17、p-STAT3蛋白相对表达量均高于空白对照组和抑制剂组(P均<0.01)。结论 IL-17A能够上调体外培养的Ha Ca T细胞K17表达,其调控机制可能是通过激活STAT3来实现,表明IL-17A在银屑病中发挥的作用可能与K17有关。展开更多
基金National Natural Science Foundation of China(Nos.81803125 and 81903192)。
文摘Background:Psoriasis is a common chronic inflammatory skin disease with 2%to 3%prevalence worldwide and a heavy social-psychological burden for patients and their families.As the exact pathogenesis of psoriasis is still unknown,the current treatment is far from satisfactory.Thus,there is an urgent need to find a more effective therapy for this disease.Keratin 17(K17),a type I intermediate filament,is overexpressed in the psoriatic epidermis and plays a critical pathogenic role by stimulating T cells in psoriasis.Therefore,we hypothesized that inhibiting K17 may be a potential therapeutic approach for psoriasis.This study aimed to investigate the therapeutic effect of K17-specific small interfering RNA(siRNA)on mice with imiquimod(IMQ)-induced psoriasis-like dermatitis.Methods:Eight-week-old female BALB/c mice were administered a 5%IMQ cream on both ears to produce psoriatic dermatitis.On day 3,K17 siRNA was mixed with an emulsion matrix and applied topically to the left ears of the mice after IMQ application every day for 7 days.The right ears of the mice were treated in parallel with negative control(NC)siRNA.Inflammation was evaluated by gross ear thickness,histopathology,the infiltration of inflammatory cells(CD3+T cells and neutrophils)using immunofluorescence,and the expression of cytokine production using real-time quantitative polymerase chain reaction.The obtained data were statistically evaluated by unpaired t-tests and a one-way analysis of variance.Results:The severity of IMQ-induced dermatitis on K17 siRNA-treated mice ears was significantly lower than that on NC siRNA-treated mice ears,as evidenced by the alleviated ear inflammation phenotype,including decreased ear thickness,infiltration of inflammatory cells(CD3+T cells and neutrophils),and inflammatory cytokine/chemokine expression levels(interleukin 17[IL-17],IL-22,IL-23,C-X-C motif chemokine ligand 1,and C-C motif chemokine ligand 20)(P<0.05 vs.the Blank or NC siRNA groups).Compared to the NC siRNA treatment,the K17 siRNA treatment resulted in increased K1 and K10 expression,which are characteristic of keratinocyte differentiation(vs.NC siRNA,K17 siRNA1 group:K1,t=4.782,P=0.0050;K10,t=3.365,P=0.0120;K17 siRNA2 group:K1,t=4.104,P=0.0093;K10,t=4.168,P=0.0042;siRNA Mix group:K1,t=3.065,P=0.0221;K10,t=10.83,P<0.0001),and decreased K16 expression,which is characteristic of keratinocyte proliferation(vs.NC siRNA,K17 siRNA1 group:t=4.156,P=0.0043;K17 siRNA2 group:t=2.834,P=0.0253;siRNA Mix group:t=2.734,P=0.0250).Conclusions:Inhibition of K17 expression by its specific siRNA significantly alleviated inflammation in mice with IMQ-induced psoriasis-like dermatitis.Thus,gene therapy targeting K17 may be a potential treatment approach for psoriasis.
文摘目的利用RNA干涉技术诱导角蛋白17(K17)基因沉默,观察其对角质形成细胞(KC)增生和凋亡等生物学活性的影响。方法合成两条含有针对人K17mRNA序列的正义和反义寡核苷酸,退火后与表达载体psilencer3.1-H1neo相连接,经鉴定后转染人角质形成细胞系HaCaT,分别以逆转录聚合酶链反应(RT-PCR)和免疫印迹法(W est-ern b lot)检测转染细胞K17 mRNA与蛋白水平的改变,用流式细胞仪检测转染细胞的细胞周期及凋亡情况,并通过透射电镜观察细胞的凋亡。结果成功构建了靶向人K17基因的siRNA表达载体psilencer3.1/K17,检测到瞬时转染的HaCaT细胞中K17的蛋白水平及mRNA水平均明显下降。流式细胞仪检测表明转染细胞的细胞周期发生了明显的G1期阻滞并证实凋亡的存在,电镜下观察到凋亡小体。结论对于增生活跃的角质形成细胞,K17的表达对其增生、分化和凋亡等生物学活性具有重要影响。靶向K17的siRNA能够抑制角质形成细胞增生,诱导其凋亡。
文摘目的观察白细胞介素-17A(IL-17A)对体外培养的人永生化角质形成细胞(Ha Ca T)角蛋白17(K17)表达及信号转导和转录激活因子3(STAT3)信号通路的影响。方法采用RPMI1640培养液培养Ha Ca T细胞,将细胞随机分为空白对照组、诱导组、抑制剂组,空白对照组仅加DMEM高糖培养基,诱导组加入含50μg/L IL-17A的DMEM高糖培养基,抑制剂组加入含50μg/L IL-17A的DMEM高糖培养基和10μmol/L STAT3抑制剂Piceatannol。收集培养的Ha Ca T细胞,采用MTT比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,RT-PCR法检测细胞K17 mRNA表达水平,Western blotting法检测细胞K17和磷酸化STAT3(p-STAT3)蛋白表达水平。结果诱导组细胞增殖A值高于空白对照组和抑制剂组,细胞凋亡率低于空白对照组和抑制剂组(P均<0.01);诱导组K17mRNA及K17、p-STAT3蛋白相对表达量均高于空白对照组和抑制剂组(P均<0.01)。结论 IL-17A能够上调体外培养的Ha Ca T细胞K17表达,其调控机制可能是通过激活STAT3来实现,表明IL-17A在银屑病中发挥的作用可能与K17有关。
