广谱caspase抑制剂抗小鼠接触性皮炎的研究
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摘要
【目的】
以Balb/c小鼠变应性接触性皮炎(ACD)模型及刺激性接触性皮炎(ICD)模型,研究外用广谱caspase抑制剂Z-VAD-FMK对皮肤炎症反应的抑制作用,观察其对T细胞的作用,阐明抗炎机制,为研制新型抗皮肤炎症外用药物提供关键数据。
【方法】
1.运用Franz垂直扩散池进行Z-VAD-FMK的体外透皮研究,高效液相色谱法(HPLC)检测药物含量;同时对其进行体内透皮研究,局部外用Z-VAD-FMK后将皮肤离体,以乙二醇去除表皮,剩余组织经液氮研磨组织匀浆,以液相色谱—串连质谱法(LC-MS/MS)检测药物浓度。
2.以1-氟-2,4-二硝基苯(DNFB)制作经典ACD小鼠模型,取激发后不同时间点对经典ACD小鼠模型的评价指标进行探讨。
3.以DNFB制作初次ACD小鼠模型,数显游标卡尺测量耳肿度随时间的动态变化,ELISA方法检测耳组织内Th1细胞因子IL-2、INF-γ及Th2细胞因子IL-4的表达水平,Real time RT-PCR方法检测细胞因子mRNA水平,流式细胞术检测耳引流淋巴结中T淋巴细胞的增殖活性及表面活化标记,从上述方面对初次ACD小鼠模型与经典ACD小鼠模型进行评价比较。
4.以经典ACD小鼠模型及初次ACD模型,观察外用Z-VAD-FMK对ACD的治疗作用、对ACD激发的抑制作用及对ACD诱导的抑制作用。
5.以ELISA方法检测Z-VAD-FMK干预ACD治疗、ACD激发及ACD诱导实验中耳组织内Th1细胞因子IL-2与INF-γ的表达水平,以real-time RT-PCR方法检测耳组织内IL-2 mRNA与INF-γmRNA的水平。
6.进行小鼠局部淋巴结分析(LLNA),以BrdU—流式细胞术检测外用Z-VAD-FMK对耳引流淋巴结中T淋巴细胞增殖活性的影响,以流式细胞术检测其对T淋巴细胞活化的影响;
7.以巴豆油制作小鼠ICD模型,观察外用Z-VAD-FMK对ICD的抑制作用。
【结果】
1.体外透皮研究中,在Franz垂直扩散池供给池为20mM Z-VAD-FMK 200μl、接收池为4ml 0.9%NaCl、有效扩散面积为1cm~2的条件下,Z-VAD-FMK 6h、12h及24h的透皮率分别为5.35%、15.52%及22.76%;体内透皮研究中,间隔12h的2次外用2.5mM Z-VAD-FMK 15μl后,其在耳组织中的浓度可达100μM,这与体外实验中的有效浓度相近。
2.经典ACD中,与激发后耳肿度一样,激发后双侧耳重量之差与激发后组织切片显微镜下同一位置左右侧耳双面距离之差这2种客观性更强的指标亦显示出一致的随时间变化趋势,即:激发后(1-2)d,炎症程度达到高峰,随后逐渐减弱,4d后已减至一半左右。
3.初次ACD小鼠模型耳背部用药6d后出现较明显的耳肿度变化,这与经典ACD模型自诱导至炎症出现的时间相同;与经典ACD小鼠模型相似,初次ACD模型的耳肿度亦呈现出随时间变化的趋势:用药后(7-8)d,耳肿度达到高峰,随后逐渐减弱,9d后已减至一半左右;二者耳组织中的浸润细胞均以单一核细胞为主;与正常对照相比,二者耳组织中Th1型细胞因子IL-2与INF-γ的表达均增加,其差别均有统计学意义,而Th2型细胞因子IL-4均未增加,差别无统计学意义;与正常对照相比,二者耳引流淋巴结中T淋巴细胞掺入的BrdU的荧光强度均增高,T淋巴细胞3种表面活化标记(CD69、CD25及Ia)阳性细胞的百分率亦均升高,其差别均有统计学意义。
4.将Z-VAD-FMK外用于ACD小鼠模型,与阴性对照相比,激发后2h用药、激发前用药及诱导前用药这3种情况下ACD观察指标均下降,其差别均有统计学意义。
5.上述3种情况下,与阴性对照相比,Z-VAD-FMK处理组耳组织中IL-2与INF-γ的表达均减少,差别均有统计学意义。
6.LLNA中,与阴性对照相比,Z-VAD-FMK处理组耳引流淋巴结中T淋巴细胞掺入的BrdU的荧光强度均减弱,T细胞3种表面活化标记(CD69、CD25及Ia)阳性细胞的百分率亦均下降,其差别均有统计学意义。
7.在干预ICD的实验中,无论是治疗研究还是预防研究,与阴性对照相比,Z-VAD-FMK处理组3个观察指标的差别均无统计学意义。
【结论】
1.广谱caspase抑制剂Z-VAD-FMK对皮肤有一定的通透性,为其作为皮肤外用药物提供了可能。
2.经典ACD小鼠模型与初次ACD小鼠模型均为Th1型免疫反应,二者在临床、组织病理学、组织中细胞因子表达及局部引流淋巴结中的表现均相似,可以互用,从而丰富了ACD小鼠模型,为研究抗炎、抗免疫新药提供了新的模型选择。
3.外用广谱caspase抑制剂Z-VAD-FMK可以有效抑制ACD的发生,其对ACD的诱导及激发亦均有抑制作用。
4.外用广谱caspase抑制剂Z-VAD-FMK对巴豆油引起的ICD无明显抑制作用,提示其抗刺激性炎症的作用较弱。
5.外用广谱caspase抑制剂Z-VAD-FMK可以抑制皮肤及局部引流淋巴结中T淋巴细胞的增殖与活化,从而有效抑制ACD的发生。
6.为了能更好地利用广谱caspase抑制剂Z-VAD-FMK局部外用的抗炎作用,需进行试剂的进一步研制,使其尽可能摆脱DMSO的影响,并对其剂型进行优化,增强透皮作用,从而将其抗皮肤炎症作用发挥到最大。
Objective
To investigate the effect of Z-VAD-FMK,a broad-spectrum caspase inhibitor,on mouse allergic contact dermatitis(ACD) and mouse irritant contact dermatitis(ICD) and study its effect on T lymphocytes.To interpret its anti-inflammatory mechanism and provide important data for developing new anti-dermatitis drugs.
Methods
1.The in vitro permeation was evaluated using Franz diffusion cells fitted with normal mouse skin and high pressure liquid chromatography(HPLC) was used to detect drug concentration.As to in vivo permeation,20%ethylene alcohol was used to remove epidermis of mouse ears on which Z-VAD-FMK was painted and then ears were grinded in liquid nitrogen.The drug concentration was determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).
2.1-Fluoro-2,4-dinitrobenzene(DNFB) was used to produce classical ACD mouse model.At different time after elicitation,the scoring system was evaluated. Several parameters were detected,including ear swelling,difference of ear weight between different side,and thickness of ear tissue under microscopic observation.
3.DNFB was used to produce primary ACD mouse model.Two models were compared with each other in three aspects.Firstly,ear swelling was detected by a digital vernier caliper and used as clinical index.Secondly,levels of T lymphocyte cytokines and their mRNA in ear tissues were detected using ELISA and real time RT-PCR respectively.In the last place,local lymph node assay(LLNA) was carried out and flow cytometry was used to detect proliferation and activation of T lymphocytes in local lymph node.
4.Classical ACD mouse model and primary ACD mouse model were used to investigate the effect of Z-VAD-FMK on ACD in three aspects:therapeutical effect,preventive effect on elicitation,and preventive effect on induction.
5.ELISA was used to detect levels of Th1 cytokines(INF-γand IL-2) in ear tissues in investigating the effect of Z-VAD-FMK on mouse ACD.Real time RT-PCR was used to detect levels of their mRNA.
6.Local lymph node assay(LLNA) was carried out.BrdU-flow cytometry was used to detect proliferation of T lymphocytes in local lymph node and flow cytometry to detect activation of T lymphocytes after topical use of Z-VAD-FMK.
7.Croton oil was used to produce ICD mouse model.To investigate its effect on ICD,Z-VAD-FMK was externally applied on the surface of the ear.
Results
1.In vitro permeation rate of Z-VAD-FMK in 6h,12h and 24h were 5.35%,15.52, and 22.76%respectively,with 200μl of 20mmol/1 Z-VAD-FMK in donor compartment,4ml of 0.9%saline in receptor,and a diffusion area of lcm~2.As to in vivo permeation,the concentration of Z-VAD-FMK in ear tissue achieved 100μmol/1 after twice topical use of 15μl of 2.5mmol/1 Z-VAD-FMK with the interval of 12h,which is similar to the effective concentration in vitro.
2.In classical ACD mouse model,similar to ear swelling,difference of ear weight between different side and thickness of ear tissue under microscopic observation also indicate that the degree of inflammation went up to the peak(1-2)d after elicitation.Then the degree of inflammation declined gradually,and reached about half of the peak at day 4.
3.In primary ACD mouse model,ear swelling response was observed 6 days after ear painting of DNFB,which was the same as the time interval from back elicitation to occurrence of ear inflammation in classical ACD.Kinetics of the inflammatory response to DNFB during primary ACD was just like that during classical ACD.Infiltrating cells in ear tissues were mostly mononuclear cells in the two models.Levels of IL-2 and INF-γwere all significantly higher in both models,compared with that in normal control,but there was no significant difference in the level of IL-4.Proliferation and activation of T lymphocytes in local lymph node was observed in both models,but not in normal control.The difference between them was significant.
4.Z-VAD-FMK was externally used on mouse ACD at three time points:2h after elicitation,before elicitation,and before induction.Results showed that in these three conditions,three evaluation indexes of Z-VAD-FMK-treated mice were all significantly lower than that of vehicle-treated mice.
5.In the above three conditions,levels of both INF-γand IL-2 in ear tissues of Z-VAD-FMK-treated mice were significantly lower than that of vehicle-treated mice.
6.In LLNA,mean intensity of BrdU in T lymphocytes of Z-VAD-FMK-treated mice was significantly weaker than that of vehicle-treated mice,and percents of activation markers-positive T lymphocytes were all significant lower than that of vehicle-treated mice too.
7.In investigation of the therapeutic effect and preventive effect of Z-VAD-FMK on ICD,there was no significant difference between Z-VAD-FMK-treated mice and vehicle-treated mice at all.
Conclusions
1.Z-VAD-FMK can permeate through mouse skin,which provides possibility to its clinical use as topical medication.
2.Just as classical ACD,primary ACD is also a type of Th1 reaction.They can take ??the place of each other in application,providing new choice for investigation of new anti-inflammatory and anti-immune drugs.
3.Topical application of Z-VAD-FMK can effectively inhibit allergic contact dermatitis in three aspects:treating,preventing induction,and preventing elicitation.
4.Z-VAD-FMK failed to inhibit ICD,which suggests that Z-VAD-FMK produces a marked effect mainly by anti-immune response;its anti-irritant inflammation effect is weaker however.
5.Topical use of Z-VAD-FMK can inhibit the activation and proliferation of T lymphocytes in both skin tissue and local lymph node,leading to the inhibitory effect of contact allergic reaction.
6.In order to bring the anti-inflammatory effect of Z-VAD-FMK into full play,we should pay more efforts to develop its dosage form and molecular structure.
以Balb/c小鼠变应性接触性皮炎(ACD)模型及刺激性接触性皮炎(ICD)模型,研究外用广谱caspase抑制剂Z-VAD-FMK对皮肤炎症反应的抑制作用,观察其对T细胞的作用,阐明抗炎机制,为研制新型抗皮肤炎症外用药物提供关键数据。
【方法】
1.运用Franz垂直扩散池进行Z-VAD-FMK的体外透皮研究,高效液相色谱法(HPLC)检测药物含量;同时对其进行体内透皮研究,局部外用Z-VAD-FMK后将皮肤离体,以乙二醇去除表皮,剩余组织经液氮研磨组织匀浆,以液相色谱—串连质谱法(LC-MS/MS)检测药物浓度。
2.以1-氟-2,4-二硝基苯(DNFB)制作经典ACD小鼠模型,取激发后不同时间点对经典ACD小鼠模型的评价指标进行探讨。
3.以DNFB制作初次ACD小鼠模型,数显游标卡尺测量耳肿度随时间的动态变化,ELISA方法检测耳组织内Th1细胞因子IL-2、INF-γ及Th2细胞因子IL-4的表达水平,Real time RT-PCR方法检测细胞因子mRNA水平,流式细胞术检测耳引流淋巴结中T淋巴细胞的增殖活性及表面活化标记,从上述方面对初次ACD小鼠模型与经典ACD小鼠模型进行评价比较。
4.以经典ACD小鼠模型及初次ACD模型,观察外用Z-VAD-FMK对ACD的治疗作用、对ACD激发的抑制作用及对ACD诱导的抑制作用。
5.以ELISA方法检测Z-VAD-FMK干预ACD治疗、ACD激发及ACD诱导实验中耳组织内Th1细胞因子IL-2与INF-γ的表达水平,以real-time RT-PCR方法检测耳组织内IL-2 mRNA与INF-γmRNA的水平。
6.进行小鼠局部淋巴结分析(LLNA),以BrdU—流式细胞术检测外用Z-VAD-FMK对耳引流淋巴结中T淋巴细胞增殖活性的影响,以流式细胞术检测其对T淋巴细胞活化的影响;
7.以巴豆油制作小鼠ICD模型,观察外用Z-VAD-FMK对ICD的抑制作用。
【结果】
1.体外透皮研究中,在Franz垂直扩散池供给池为20mM Z-VAD-FMK 200μl、接收池为4ml 0.9%NaCl、有效扩散面积为1cm~2的条件下,Z-VAD-FMK 6h、12h及24h的透皮率分别为5.35%、15.52%及22.76%;体内透皮研究中,间隔12h的2次外用2.5mM Z-VAD-FMK 15μl后,其在耳组织中的浓度可达100μM,这与体外实验中的有效浓度相近。
2.经典ACD中,与激发后耳肿度一样,激发后双侧耳重量之差与激发后组织切片显微镜下同一位置左右侧耳双面距离之差这2种客观性更强的指标亦显示出一致的随时间变化趋势,即:激发后(1-2)d,炎症程度达到高峰,随后逐渐减弱,4d后已减至一半左右。
3.初次ACD小鼠模型耳背部用药6d后出现较明显的耳肿度变化,这与经典ACD模型自诱导至炎症出现的时间相同;与经典ACD小鼠模型相似,初次ACD模型的耳肿度亦呈现出随时间变化的趋势:用药后(7-8)d,耳肿度达到高峰,随后逐渐减弱,9d后已减至一半左右;二者耳组织中的浸润细胞均以单一核细胞为主;与正常对照相比,二者耳组织中Th1型细胞因子IL-2与INF-γ的表达均增加,其差别均有统计学意义,而Th2型细胞因子IL-4均未增加,差别无统计学意义;与正常对照相比,二者耳引流淋巴结中T淋巴细胞掺入的BrdU的荧光强度均增高,T淋巴细胞3种表面活化标记(CD69、CD25及Ia)阳性细胞的百分率亦均升高,其差别均有统计学意义。
4.将Z-VAD-FMK外用于ACD小鼠模型,与阴性对照相比,激发后2h用药、激发前用药及诱导前用药这3种情况下ACD观察指标均下降,其差别均有统计学意义。
5.上述3种情况下,与阴性对照相比,Z-VAD-FMK处理组耳组织中IL-2与INF-γ的表达均减少,差别均有统计学意义。
6.LLNA中,与阴性对照相比,Z-VAD-FMK处理组耳引流淋巴结中T淋巴细胞掺入的BrdU的荧光强度均减弱,T细胞3种表面活化标记(CD69、CD25及Ia)阳性细胞的百分率亦均下降,其差别均有统计学意义。
7.在干预ICD的实验中,无论是治疗研究还是预防研究,与阴性对照相比,Z-VAD-FMK处理组3个观察指标的差别均无统计学意义。
【结论】
1.广谱caspase抑制剂Z-VAD-FMK对皮肤有一定的通透性,为其作为皮肤外用药物提供了可能。
2.经典ACD小鼠模型与初次ACD小鼠模型均为Th1型免疫反应,二者在临床、组织病理学、组织中细胞因子表达及局部引流淋巴结中的表现均相似,可以互用,从而丰富了ACD小鼠模型,为研究抗炎、抗免疫新药提供了新的模型选择。
3.外用广谱caspase抑制剂Z-VAD-FMK可以有效抑制ACD的发生,其对ACD的诱导及激发亦均有抑制作用。
4.外用广谱caspase抑制剂Z-VAD-FMK对巴豆油引起的ICD无明显抑制作用,提示其抗刺激性炎症的作用较弱。
5.外用广谱caspase抑制剂Z-VAD-FMK可以抑制皮肤及局部引流淋巴结中T淋巴细胞的增殖与活化,从而有效抑制ACD的发生。
6.为了能更好地利用广谱caspase抑制剂Z-VAD-FMK局部外用的抗炎作用,需进行试剂的进一步研制,使其尽可能摆脱DMSO的影响,并对其剂型进行优化,增强透皮作用,从而将其抗皮肤炎症作用发挥到最大。
Objective
To investigate the effect of Z-VAD-FMK,a broad-spectrum caspase inhibitor,on mouse allergic contact dermatitis(ACD) and mouse irritant contact dermatitis(ICD) and study its effect on T lymphocytes.To interpret its anti-inflammatory mechanism and provide important data for developing new anti-dermatitis drugs.
Methods
1.The in vitro permeation was evaluated using Franz diffusion cells fitted with normal mouse skin and high pressure liquid chromatography(HPLC) was used to detect drug concentration.As to in vivo permeation,20%ethylene alcohol was used to remove epidermis of mouse ears on which Z-VAD-FMK was painted and then ears were grinded in liquid nitrogen.The drug concentration was determined by liquid chromatography-tandem mass spectrometry(LC-MS/MS).
2.1-Fluoro-2,4-dinitrobenzene(DNFB) was used to produce classical ACD mouse model.At different time after elicitation,the scoring system was evaluated. Several parameters were detected,including ear swelling,difference of ear weight between different side,and thickness of ear tissue under microscopic observation.
3.DNFB was used to produce primary ACD mouse model.Two models were compared with each other in three aspects.Firstly,ear swelling was detected by a digital vernier caliper and used as clinical index.Secondly,levels of T lymphocyte cytokines and their mRNA in ear tissues were detected using ELISA and real time RT-PCR respectively.In the last place,local lymph node assay(LLNA) was carried out and flow cytometry was used to detect proliferation and activation of T lymphocytes in local lymph node.
4.Classical ACD mouse model and primary ACD mouse model were used to investigate the effect of Z-VAD-FMK on ACD in three aspects:therapeutical effect,preventive effect on elicitation,and preventive effect on induction.
5.ELISA was used to detect levels of Th1 cytokines(INF-γand IL-2) in ear tissues in investigating the effect of Z-VAD-FMK on mouse ACD.Real time RT-PCR was used to detect levels of their mRNA.
6.Local lymph node assay(LLNA) was carried out.BrdU-flow cytometry was used to detect proliferation of T lymphocytes in local lymph node and flow cytometry to detect activation of T lymphocytes after topical use of Z-VAD-FMK.
7.Croton oil was used to produce ICD mouse model.To investigate its effect on ICD,Z-VAD-FMK was externally applied on the surface of the ear.
Results
1.In vitro permeation rate of Z-VAD-FMK in 6h,12h and 24h were 5.35%,15.52, and 22.76%respectively,with 200μl of 20mmol/1 Z-VAD-FMK in donor compartment,4ml of 0.9%saline in receptor,and a diffusion area of lcm~2.As to in vivo permeation,the concentration of Z-VAD-FMK in ear tissue achieved 100μmol/1 after twice topical use of 15μl of 2.5mmol/1 Z-VAD-FMK with the interval of 12h,which is similar to the effective concentration in vitro.
2.In classical ACD mouse model,similar to ear swelling,difference of ear weight between different side and thickness of ear tissue under microscopic observation also indicate that the degree of inflammation went up to the peak(1-2)d after elicitation.Then the degree of inflammation declined gradually,and reached about half of the peak at day 4.
3.In primary ACD mouse model,ear swelling response was observed 6 days after ear painting of DNFB,which was the same as the time interval from back elicitation to occurrence of ear inflammation in classical ACD.Kinetics of the inflammatory response to DNFB during primary ACD was just like that during classical ACD.Infiltrating cells in ear tissues were mostly mononuclear cells in the two models.Levels of IL-2 and INF-γwere all significantly higher in both models,compared with that in normal control,but there was no significant difference in the level of IL-4.Proliferation and activation of T lymphocytes in local lymph node was observed in both models,but not in normal control.The difference between them was significant.
4.Z-VAD-FMK was externally used on mouse ACD at three time points:2h after elicitation,before elicitation,and before induction.Results showed that in these three conditions,three evaluation indexes of Z-VAD-FMK-treated mice were all significantly lower than that of vehicle-treated mice.
5.In the above three conditions,levels of both INF-γand IL-2 in ear tissues of Z-VAD-FMK-treated mice were significantly lower than that of vehicle-treated mice.
6.In LLNA,mean intensity of BrdU in T lymphocytes of Z-VAD-FMK-treated mice was significantly weaker than that of vehicle-treated mice,and percents of activation markers-positive T lymphocytes were all significant lower than that of vehicle-treated mice too.
7.In investigation of the therapeutic effect and preventive effect of Z-VAD-FMK on ICD,there was no significant difference between Z-VAD-FMK-treated mice and vehicle-treated mice at all.
Conclusions
1.Z-VAD-FMK can permeate through mouse skin,which provides possibility to its clinical use as topical medication.
2.Just as classical ACD,primary ACD is also a type of Th1 reaction.They can take ??the place of each other in application,providing new choice for investigation of new anti-inflammatory and anti-immune drugs.
3.Topical application of Z-VAD-FMK can effectively inhibit allergic contact dermatitis in three aspects:treating,preventing induction,and preventing elicitation.
4.Z-VAD-FMK failed to inhibit ICD,which suggests that Z-VAD-FMK produces a marked effect mainly by anti-immune response;its anti-irritant inflammation effect is weaker however.
5.Topical use of Z-VAD-FMK can inhibit the activation and proliferation of T lymphocytes in both skin tissue and local lymph node,leading to the inhibitory effect of contact allergic reaction.
6.In order to bring the anti-inflammatory effect of Z-VAD-FMK into full play,we should pay more efforts to develop its dosage form and molecular structure.
引文
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4. Logue SE, Martin SJ. Caspase activation cascades in apoptosis. Biochem Soc Trans, 2008, 36(Pt 1): 1-9.
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8. Salmena L, Lemmers B, Hakem A, et al. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes Dev, 2003, 17(7): 883-895.
9. Falk M, Ussat S, Reiling N, et al. Caspase inhibition blocks human T cell proliferation by suppressing appropriate regulation of IL-2, CD25, and cell cycle-associated proteins. J Immunol, 2004, 173(8): 5077-5085.
10. Krakauer T. Caspase inhibitors attenuate superantigen-induced inflammatory cytokines, chemokines, and T-Cell proliferation. Clin Diagn Lab Immunol, 2004, 11(3): 621-624.
11.Watanabe H, Gaide O, Petrilli V, et al. Activation of the IL-1β-processing inflammasome is involved in contact hypersensitivity.J Invest Dermatol,2007,127(8):1956-1963.
12.Antonopoulos C,Cumberbatch M,Dearman RJ,et al.Functional caspase-1 is required for Langerhans cell migration and optimal contact sensitization in mice.J Immunol,2001,166(6):3672-3677.
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1. Shi, Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell, 2002, 9(3): 459-470.
2. Park K, Kuechle MK, Choe Y, et al. Expression and characterization of constitutively active human caspase-14. Biochem Biophys Res Commun, 2006, 347(4): 941-948.
3. Martinon F, Tschopp J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases. Cell, 2004, 117(5): 561-574.
4. Koenig U, Eckhart L, Tschachler E. Evidence that caspase-13 is not a human but a bovine gene. Biochem. Biophys. Res. Commun., 2001, 285(5): 1150-1154.
5. Eckhart L, Ballaun C, Uthman A, et al. Identification and characterization of a novel mammalian caspase with proapoptotic activity. J Biol Chem, 2005, 280(42): 35077-35080.
6. Kennedy NJ, Kataoka T, Tschopp J, et al. Caspase activation is required for T cell proliferation. J Exp Med, 1999, 190(12): 1891-1896.
7. Alam A, Cohen LY, Aouad S, et al. Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells. J Exp Med, 1999, 190(12): 1879-1890.
8. Falk M, Ussat S, Reiling N, et al. Caspase inhibition blocks human T cell proliferation by suppressing appropriate regulation of IL-2, CD25, and cell cycle-associated proteins. J Immunol, 2004, 173(8): 5077-5085.
9. Aouad SM, Cohen LY, Sharif-Askari E, et al. Caspase-3 is a component of fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death. J Immunol, 2004, 172(4): 2316-2323.
10. Mukerjee N, McGinnis KM, Gnegy ME, et al. Caspase-mediated calcineurin activation contributes to IL-2 release during T cell activation. Biochem Biophys Res Commun, 2001, 285(5): 1192-1199.
11. Chun HJ, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature, 2002, 419(6905): 395-399.
12. Salmena L, Lemmers B, Hakem A, et al. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes Dev, 2003, 17(7): 883-895.
13. Boissonnas A, Bonduelle O, Lucas B, et al. Differential requirement of caspases during naive T cell proliferation. Eur J Immunol, 2002, 32(10): 3007-3015.
14. Sordet O, Rebe C, Plenchette S, et al. Specific involvement of caspases in the differentiation of monocytes into macrophages. Blood, 2002, 100(13): 4446-4453.
15. Degterev A, Boyce M, Yuan J. A decade of caspases. Oncogene, 2003, 22(53): 8543-8567.
16. Ekert PG, Silke J, Vaux DL. Caspase inhibitors. Cell Death Differ, 1999, 6(11): 1081-1086.
17. Krakauer T. Caspase inhibitors attenuate superantigen-induced inflammatory cytokines, chemokines, and T-Cell proliferation. Clin Diagn Lab Immunol, 2004, 11(3): 621-624.
18. Mack A, Hacker G. Inhibition of caspase or FADD function blocks proliferation but not MAP kinase-activation and interleukin-2-production during primary stimulation of T cells. Eur J Immunol, 2002, 32(7): 1986-1992.
19. Beisner DR, Chu IH, Arechiga AF, et al. The requirements for Fas-associated death domain signaling in mature T cell activation and survival. J Immunol, 2003, 171(1): 247-256.
20. Tibbetts MD, Zheng L, Lenardo MJ. The death effector domain protein family: regulators of cellular homeostasis. Nat Immunol, 2003, 4(5): 404-409.
21. Iwata A, Nishio K, Winn RK, et al. A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model. J Immunol, 2003, 170(6): 3386-3391.
22. Algeciras-Schimnich A, Barnhart BC, Peter ME. Apoptosis-independent functions of killer caspases. Curr Opin Cell Biol, 2002, 14(6): 721-726.
2. Weischer M, Rocken M, Berneburg M. Calcineurin inhibitors and rapamycin: cancer protection or promotion? Exp Dermatol, 2007, 16: 385-393.
3. Shi, Y. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell, 2002, 9: 459-470.
4. Logue SE, Martin SJ. Caspase activation cascades in apoptosis. Biochem Soc Trans, 2008, 36(Pt 1): 1-9.
5. Kennedy NJ, Kataoka T, Tschopp J, et al. Caspase activation is required for T cell proliferation. J Exp Med, 1999, 190(12): 1891-1896.
6. Alam A, Cohen LY, Aouad S, et al. Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells. J Exp Med, 1999, 190(12): 1879-1890.
7. Chun HJ, Zheng L, Ahmad M, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature, 2002, 419(6905): 395-399.
8. Salmena L, Lemmers B, Hakem A, et al. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes Dev, 2003, 17(7): 883-895.
9. Falk M, Ussat S, Reiling N, et al. Caspase inhibition blocks human T cell proliferation by suppressing appropriate regulation of IL-2, CD25, and cell cycle-associated proteins. J Immunol, 2004, 173(8): 5077-5085.
10. Krakauer T. Caspase inhibitors attenuate superantigen-induced inflammatory cytokines, chemokines, and T-Cell proliferation. Clin Diagn Lab Immunol, 2004, 11(3): 621-624.
11.Watanabe H, Gaide O, Petrilli V, et al. Activation of the IL-1β-processing inflammasome is involved in contact hypersensitivity.J Invest Dermatol,2007,127(8):1956-1963.
12.Antonopoulos C,Cumberbatch M,Dearman RJ,et al.Functional caspase-1 is required for Langerhans cell migration and optimal contact sensitization in mice.J Immunol,2001,166(6):3672-3677.
13.Iwata A,Nishio K,Winn RK,et al.A broad-spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model.J Immunol,2003,170(6):3386-3391.
14.Yamaguchi K,Mitsui T,Aso Y,et al.Structure-permeability relationship analysis of the permeation barrier properties of the stratum corneum and viable epidermis/dermis of rat skin.J Pharm Sci,2008,Jan 28[Epub ahead of print].
15.Kao J,Hall J,Helman G.In vitro percutaneous absorption in mouse skin:influence of skin appendages.Toxicol Appl Pharmacol,1988,94(1):93-103.
16.崔福德,潘卫三,毛世瑞,等.药剂学.北京:中国医药科技出版社,2002:553-555.
17.Ekert PG,Silke J,Vaux DL.Caspase inhibitors.Cell Death Differ,1999,6(11):1081-1086.
18.Savic S,Savic M,Tamburic S,et al.An alkylpolyglucoside surfactant as a prospective pharmaceutical excipient for topical formulations:the influence of oil polarity on the colloidal structure and hydrocortisone in vitro/in vivo permeation.Eur J Pharm Sci,2007,30(5):441-450.
19.Puglia C,Blasi P,Rizza L,et al.Lipid nanoparticles for prolonged topical delivery:An in vitro and in vivo investigation.Int J Pharm,2008 Feb 3[Epub ahead of print].
20.Brown PR,Krstulovic AM,Hartwick RA.Current state of the art in the HPLC analyses of free nucleotides,nucleosides,and bases in biological fluids.Adv Chromatogr,1980,18:101-138.
21.Callesen AK,Vach W,Jorgensen PE,et al.Reproducibility of Mass Spectrometry Based Protein Profiles for Diagnosis of Breast Cancer across Clinical Studies:A Systematic Review. J Proteome Res, 2008, 7(4): 1395-1402.
22. Wang S, Wang XH. Analytical methods for the determination of zeranol residues in animal products: A review. Food Addit Contam, 2007, 24(6): 573-582.
23. Holcapek M, Kolarova L, Nobilis M. High-performance liquid chromatography-tandem mass spectrometry in the identification and determination of phase I and phase II drug metabolites. Anal Bioanal Chem, 2008 Mar 15; [Epub ahead of print].
24. Scholzen TE, Stander S, Riemann H, et al. Modulation of cutaneous inflammation by angiotensin-converting enzyme. J Immunol, 2003, 170(7): 3866-3873.
25. Saint-Mezard P, Krasteva M, Chavagnac C, et al. Afferent and efferent phases of allergic contact dermatitis (ACD) can be induced after a single skin contact with haptens: evidence using a mouse model of primary ACD. J Invest Dermatol, 2003, 120(4): 641-647.
26. van Houwelingen AH, Kaczynska K, Kraneveld AD, et al. Topical application of F991, an immunoglobulin free light chain antagonist, prevents development of contact sensitivity in mice. Clin Exp Allergy, 2007, 37(2): 270-275.
27. Harada D, Takada C, Tsukumo Y, et al. Analyses of a mouse model of the dermatitis caused by 2,4,6-trinitro-1-chlorobenzene(TNCB)-repeated application. J Dermatol Sci, 2005, 37(3): 159-167.
28. Wang B, Fujisawa H, Zhuang L, et al. CD4+ Th1 and CD8+ type 1 cytotoxic T cells both play a crucial role in the full development of contact hypersensitivity. J Immunol, 2000, 165(12): 6783-6790.
29. Girardi M., Sherling MA, Filler RB, et al. Anti-inflammatory effects in the skin of thymosin-beta4 splice-variants. Immunology, 2003, 109(1): 1-7.
30. Bhol KC, Schechter PJ. Topical nanocrystalline silver cream suppresses inflammatory cytokines and induces apoptosis of inflammatory cells in a murine model of allergic contact dermatitis. Br J Dermatol, 2005, 152(6): 1235-1242.
31.Wille JJ, Kydonieus A, Kalish RS. Inhibition of irritation and contact hypersensitivity by phenoxyacetic acid methyl ester in mice. Skin Pharmacol Appl Skin Physiol, 2000, 13(2): 65-74.
32. Baumer W, Seegers U, Braun M, et al. TARC and RANTES, but not CTACK, are induced in two models of allergic contact dermatitis. Effects of cilomilast and diflorasone diacetate on T-cell-attracting chemokines. Br J Dermatol, 2004, 151(4): 323-830.
33. Baumer W, Krekeler S, DeVries VC, et al. Non-steroidal and steroidal anti-inflammatory drugs vary in their modulation of dendritic cell function in the elicitation phase of allergic contact dermatitis. Exp Dermatol, 2006, 15(4): 322-329.
34. Igawa K, Yokozeki H, Miyazaki Y, et al. Topical glucocorticoids application induced an augmentation in the expression of IL-1 alpha while inhibiting the expression of IL-10 in the epidermis in murine contact hypersensitivity. Clin Exp Allergy, 2001, 31(3): 485-494.
35. Takaoka A, Arai I, Sugimoto M, et al. Involvement of IL-31 on scratching behavior in NC/Nga mice with atopic-like dermatitis. Exp Dermatol, 2006, 15(3): 161-167.
36. Nuriya S, Enomoto S, Azuma M. The role of CTLA-4 in murine contact hypersensitivity. J Invest Dermatol, 2001, 116(5) :764-768.
37. Waltz SE, Eaton L, Toney-Earley K, et al. Ron-mediated cytoplasmic signaling is dispensable for viability but is required to limit inflammatory responses. J Clin Invest, 2001, 108(4): 567-576.
38. Garside R, Stein K, Castelnuovo E, et al. The effectiveness and cost-effectiveness of pimecrolimus and tacrolimus for atopic eczema: a systematic review and economic evaluation. Health Technol Assess, 2005, 9(29):iii, xi-xiii, 1-230.
39. Kolarz B, Targonska-Stepniak B, Darmochwal-Kolarz D, et al. Autoimmune aspects of treatment with TNF-alpha inhibitors. Postepy Hig Med Dosw (Online), 2007,61:478-484.
40. Park K, Kuechle MK, Choe Y, et al. Expression and characterization of constitutively active human caspase-14. Biochem Biophys Res Commun, 2006, 347(4): 941-948.
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