摘要
第一部分Toll样受体2及相关因子在银屑病患者皮损中的表达目的:探讨TLR2、信号途径下游分子NF-κBp65,以及可能的效应分子TGF-α在银屑病患者皮损中的表达及其与银屑病严重程度的关系。方法:采用EliVision免疫组化法检测40例银屑病患者进行期皮损、11例非皮损区及15例正常人皮肤中TLR2、NF-κBp65、TGF-α的表达,对其在皮损中的表达进行相关性分析,并将结果与PASI评分进行相关性分析。结果:与正常人皮肤及银屑病患者非皮损区相比,银屑病患者皮损表皮中TLR2、NF-κBp65、TGF-α的表达明显上调,而非皮损区TLR2的表达也高于正常人皮肤,差异均有统计学意义(P＜0.05)。银屑病患者皮损表皮中TLR2、NF-κBp65的表达水平与PASI评分之间存在正相关(P＜0.05)。银屑病患者皮损表皮中TLR2与NF-κBp65、TLR2与TGF-α、NF-κBp65与TGF-α表达水平之间均存在正相关(P＜0.05)。结论:TLR2、NF-κBp65、TGF-α在银屑病中表达异常,可能共同参与了银屑病的发病过程。
第二部分Toll样受体2对人角质形成细胞增殖的影响
目的:探讨TLR2对体外培养的人角质形成细胞增殖的影响。方法:应用天然配体肽聚糖(PGN)体外活化人角质形成细胞TLR2,用MTT法检测PGN对角质形成细胞体外增殖的影响,确定最适作用浓度;用实时荧光定量PCR法和Western-blot方法分别检测角质形成细胞TLR2、细胞增殖标记Ki67基因转录和蛋白的表达水平;采用抗体封闭实验分析封闭TLR2对PGN诱导角质形成细胞表达TLR2、Ki67的影响。结果:不同浓度的PGN处理角质形成细胞后24小时,在1.25、2.5、5ug/mL的浓度下细胞出现明显增殖(P＜0.05)。1.25ug/mL PGN作用后12小时和24小时角质形成细胞TLR2 mRNA的表达明显增高,24小时和48小时Ki67 mRNA的表达明显增高;1.25、2.5和5ug/mL PGN作用后24小时,各浓度组角质形成细胞TLR2 mRNA和蛋白的表达均增加,其中1.25和2.5ug/mL浓度下Ki67 mRNA的表达明显增加,各浓度组Ki67蛋白合成均增加(P＜0.05)。以抗人TLR2单克隆阻断性抗体封闭TLR2后,PGN诱导的角质形成细胞TLR2、Ki67 mRNA和蛋白表达的上调均受到明显抑制(P＜0.05)。结论:角质形成细胞TLR2信号途径的活化可促进角质形成细胞的增殖,这一过程可能参与了银屑病的发病。
第三部分Toll样受体2在人角质形成细胞增殖中的作用机制探讨
目的:研究TLR2对体外培养的人角质形成细胞NF-κB活化及TGF-α表达的影响,以探讨TLR2在人角质形成细胞增殖中的可能作用机制。方法:应用天然配体肽聚糖(PGN)体外活化人角质形成细胞TLR2,用实时荧光定量PCR法检测角质形成细胞NF-κBp65、TGF-αmRNA的表达,用Western-blot方法检测核内NF-κBp65和细胞TGF-α蛋白的表达;采用抗体封闭实验分析封闭TLR2对PGN诱导角质形成细胞NF-κB活化和TGF-α表达的影响。结果:1.25ug/mLPGN作用后24小时角质形成细胞NF-κBp65 mRNA的表达明显增高,6小时和24小时TGF-αmRNA的表达明显增高;1.25、2.5和5ug/mL PGN作用后24小时,各浓度组角质形成细胞核内NF-κBp65蛋白合成均增加,其中1.25和5ug/mL浓度下TGF-αmRNA和蛋白表达均明显增加(P＜0.05)。以抗人TLR2单克隆阻断性抗体封闭TLR2后,PGN诱导的角质形成细胞NF-κB活化及TGF-α表达上调均受到明显抑制(P＜0.05)。结论:角质形成细胞TLR2经PGN诱导活化后,可能通过促进NF-κB活化及TGF-α表达而导致角质形成细胞的异常增殖。
PartⅠThe expressions of Toll-like receptor 2 and its related factors in psoriatic lesions
Objective To investigate the expressions of Toll-like receptor 2(TLR2), nuclear-factor-κB(NF-κB) p65 and transforming growth factor-alpha(TGF-alpha) in the epidemis of psoriasis and their correlation with disease severity in psoriatic patients.Methods Forty samples of lesional psoriatic skin,eleven samples of nonlesional psoriatic skin and fifteen samples of normal skin were analyzed by EliVision immunohistochemical technique for the expressions of TLR2,NF-κBp65 and TGF-alpha protein.Correlation analysis was performed among the expressions of three factors as well as between the expressions and disease severity.Results Compared with normal epidermis and nonlesional psoriatic epidermis,TLR2,NF-κB p65 and TGF-alpha were more highly expressed in lesional epidermis from patients with psoriasis(all P＜0.05).In nonlesional psoriatic epidermis,TLR2 expression was also upregulated compared with normal epidermis(P＜0.05).The expressions of TLR2 and NF-κBp65 in lesional epidermis positively correlated individually with the psoriasis area and severity index(PASI) scores in patients(both P＜0.05).Positive correlation was also found between the expressions of any two factors(TLR2,NF-κB p65 or TGF-alpha) in lesional epidermis(all P＜0.05).Conclusion The upregulation of TLR2,NF-κBp65 and TGF-alpha expressions in psoriatic lesions may be involved in the pathogenesis of psoriasis.
PartⅡEffect of Toll-like receptor 2 on the proliferation of human keratinocytes
Objective To investigate the effect of TLR2 on the proliferation of human keratinocytes.Methods Keratinocytes were isolated from the foreskin of children, and subjected to primary culture.After 3-5 passages,the keratinocytes were incubated with peptidoglycan(PGN),a TLR2 agonist.Cell proliferation was detected by MTT colorimetric assay and the suitable concentrations of PGN were determined.The mRNA and protein expressions of TLR2 and Ki67 were detected by real-time quantitative PCR and Western blot.Antibody blocking test was utilized to evaluate the effect on TLR2 and Ki67 expressions of keratinocytes by blocking TLR2 with specific anti-TLR2 neutralizing monoclonal antibody before incubation with PGN.
Results The keratinocytes proliferation was promoted by the incubation with PGN of 1.25,2.5 and 5ug/mL for 24 hours(all P＜0.05).1.25ug/mL PGN up-regulated TLR2 mRNA expression at 12h and 24h after the incubation,and Ki67 mRNA expression at 24h and 48h.In the keratinocytes incubated with PGN of 1.25,2.5 and 5ug/mL for 24 h,TLR2 mRNA and protein,Ki67 protein were all more highly expressed in different groups,and Ki67 mRNA expression was upregulated at the concentrations of 1.25 and 2.5ug/mL(all P＜0.05).The mRNA and protein expressions of TLR2 and Ki67 were all inhibited by blocking TLR2 when incubation with PGN(all P＜0.05).
Conclusion Activation of TLR2 by PGN could promote the in vitro proliferation of cultured human keratinocytes,which provide supporting evidence for the role of TLR2 in psoriasis.
PartⅢStudy on the mechanism of Toll-like receptor 2 in promoting human keratinocytes proliferation
Objective To evaluate the effect of TLR2 on NF-κBp65 activation and TGF-alpha expression in human keratinocytes.Methods Cultured keratinocytes were incubated with peptidoglycan(PGN).Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expressions ofNF-κBp65 and TGF-alpha,respectively. Antibody blocking test was utilized to evaluate the effect on NF-κBp65 activation and TGF-alpha expression of keratinocytes by blocking TLR2 with specific anti-TLR2 neutralizing monoclonal antibodie before incubation with PGN.Results 1.25ug/mL PGN up-regulated NF-κBp65 mRNA expression in keratinocytes at 24h after the incubation,and TGF-αmRNA expression at 6h and 24h.In the keratinocytes incubated with PGN of 1.25,2.5 and 5ug/mL for 24 h,nuclear NF-κB p65 protein was more highly expressed in different groups,and TGF-αmRNA and protein expressions were upregulated at the concentrations of 1.25 and 5ug/mL(all P＜0.05). NF-κB p65 activation and TGF-alpha expression were both inhibited by blocking TLR2 when incubation with PGN(all P＜0.05).Conclusion Activation of TLR2 by PGN could promote NF-kappaB activation and TGF-αexpression,which may be one of the mechanisms of human keratinocytes proliferation.
引文
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