CAJ | 학술논문

目的研究外用广谱半胱氨酸天冬氨酸蛋白酶(caspase)抑制剂苄氧羰基-缬氨酰-丙氨酰-门冬氨酰氟甲基酮(Z-VAD-FMK)对小鼠变应性接触性皮炎(ACD)激发阶段的抑制作用,探讨其对T细胞的作用.方法以2,4二硝基氟苯(DNFB)制作小鼠经典ACD模型,于激发前外用不同浓度的Z-VAD-FMK,以耳肿度、双耳重量之差及组织切片中同一位置双耳双面距离之差为指标,观察Z-VAD-FMK对小鼠ACD激发阶段的抑制作用；以酶联免疫吸附法(ELISA)检测小鼠耳皮损中T细胞因子干扰素γ(INF-γ)及白细胞介素2(IL-2)的含量,以实时反转录PCR( real-time RT-PCR)方法检测上述因子mRNA水平(实验结果以“相对于每百万管家基因的拷贝数”表示)；进行局部淋巴结分析(LLNA),以5-溴脱氧尿核苷(BrdU)-流式细胞术检测耳引流淋巴结中淋巴细胞增殖活性,以流式细胞术检测淋巴细胞表面活化标记.结果 1.25 mmol/L Z-VAD-FMK组小鼠耳肿度为(12.6±1.2)×10-2 mm,双耳重量差为(3.1±0.2)mg,镜下双耳双面距离差为(12.1±1.1)×10-2 mm,均低于阴性对照组[(17.4±1.6)× 10-2mm,(4.2±0.3) mg,(16.7±1.5)×10-2mm;q =3.25、2.98、3.12,均P＜0.05].1.25 mmol/L Z-VAD-FMK组小鼠耳皮损中INF-γ与IL-2mRNA及蛋白的表达均低于阴性对照组[ INF-γ mRNA:152±12比220±15,IL-2 mRNA:96 ±8比156±11,q=3.15、3.42; INF-γ蛋白:(856±45) pg/ml比(1180±58)pg/ml,IL-2蛋白:(167±12) pg/ml比(225±16) pg/ml,q=3.11、3.14；均P＜0.05].1.25 mmol/L Z-VAD-FMK组小鼠耳引流淋巴结中T细胞内掺入的BrdU的平均荧光强度明显低于阴性对照组(185±15比298±21,q=3.02,P＜0.05),T细胞3种表面活化标记CD69、CD25与Ia阳性细胞的百分率亦均明显低于阴性对照组(7.8％±0.7％比10.5％±1.0％,9.8％±0.8％比14.5％±1.1％,31.2％±2.8％比46.5％±3.2％,q =3.16、3.52、3.11,均P＜0.05).结论在小鼠ACD激发前外用广谱caspase抑制剂Z-VAD-FMK可以抑制小鼠皮损局部及耳引流淋巴结中T细胞的增殖与活化,从而抑制小鼠ACD的发生. 目的研究外用廣譜半胱氨痠天鼕氨痠蛋白酶(caspase)抑製劑芐氧羰基-纈氨酰-丙氨酰-門鼕氨酰氟甲基酮(Z-VAD-FMK)對小鼠變應性接觸性皮炎(ACD)激髮階段的抑製作用,探討其對T細胞的作用.方法以2,4二硝基氟苯(DNFB)製作小鼠經典ACD模型,于激髮前外用不同濃度的Z-VAD-FMK,以耳腫度、雙耳重量之差及組織切片中同一位置雙耳雙麵距離之差為指標,觀察Z-VAD-FMK對小鼠ACD激髮階段的抑製作用；以酶聯免疫吸附法(ELISA)檢測小鼠耳皮損中T細胞因子榦擾素γ(INF-γ)及白細胞介素2(IL-2)的含量,以實時反轉錄PCR( real-time RT-PCR)方法檢測上述因子mRNA水平(實驗結果以“相對于每百萬管傢基因的拷貝數”錶示)；進行跼部淋巴結分析(LLNA),以5-溴脫氧尿覈苷(BrdU)-流式細胞術檢測耳引流淋巴結中淋巴細胞增殖活性,以流式細胞術檢測淋巴細胞錶麵活化標記.結果 1.25 mmol/L Z-VAD-FMK組小鼠耳腫度為(12.6±1.2)×10-2 mm,雙耳重量差為(3.1±0.2)mg,鏡下雙耳雙麵距離差為(12.1±1.1)×10-2 mm,均低于陰性對照組[(17.4±1.6)× 10-2mm,(4.2±0.3) mg,(16.7±1.5)×10-2mm;q =3.25、2.98、3.12,均P＜0.05].1.25 mmol/L Z-VAD-FMK組小鼠耳皮損中INF-γ與IL-2mRNA及蛋白的錶達均低于陰性對照組[ INF-γ mRNA:152±12比220±15,IL-2 mRNA:96 ±8比156±11,q=3.15、3.42; INF-γ蛋白:(856±45) pg/ml比(1180±58)pg/ml,IL-2蛋白:(167±12) pg/ml比(225±16) pg/ml,q=3.11、3.14；均P＜0.05].1.25 mmol/L Z-VAD-FMK組小鼠耳引流淋巴結中T細胞內摻入的BrdU的平均熒光彊度明顯低于陰性對照組(185±15比298±21,q=3.02,P＜0.05),T細胞3種錶麵活化標記CD69、CD25與Ia暘性細胞的百分率亦均明顯低于陰性對照組(7.8％±0.7％比10.5％±1.0％,9.8％±0.8％比14.5％±1.1％,31.2％±2.8％比46.5％±3.2％,q =3.16、3.52、3.11,均P＜0.05).結論在小鼠ACD激髮前外用廣譜caspase抑製劑Z-VAD-FMK可以抑製小鼠皮損跼部及耳引流淋巴結中T細胞的增殖與活化,從而抑製小鼠ACD的髮生.
목적 연구외용엄보반광안산천동안산단백매(caspase)억제제변양탄기-힐안선-병안선-문동안선불갑기동(Z-VAD-FMK)대소서변응성접촉성피염(ACD)격발계단적억제작용,탐토기대T세포적작용.방법 이2,4이초기불분(DNFB)제작소서경전ACD모형,우격발전외용불동농도적Z-VAD-FMK,이이종도、쌍이중량지차급조직절편중동일위치쌍이쌍면거리지차위지표,관찰Z-VAD-FMK대소서ACD격발계단적억제작용；이매련면역흡부법(ELISA)검측소서이피손중T세포인자간우소γ(INF-γ)급백세포개소2(IL-2)적함량,이실시반전록PCR( real-time RT-PCR)방법검측상술인자mRNA수평(실험결과이“상대우매백만관가기인적고패수”표시)；진행국부림파결분석(LLNA),이5-추탈양뇨핵감(BrdU)-류식세포술검측이인류림파결중림파세포증식활성,이류식세포술검측림파세포표면활화표기.결과 1.25 mmol/L Z-VAD-FMK조소서이종도위(12.6±1.2)×10-2 mm,쌍이중량차위(3.1±0.2)mg,경하쌍이쌍면거리차위(12.1±1.1)×10-2 mm,균저우음성대조조[(17.4±1.6)× 10-2mm,(4.2±0.3) mg,(16.7±1.5)×10-2mm;q =3.25、2.98、3.12,균P＜0.05].1.25 mmol/L Z-VAD-FMK조소서이피손중INF-γ여IL-2mRNA급단백적표체균저우음성대조조[ INF-γ mRNA:152±12비220±15,IL-2 mRNA:96 ±8비156±11,q=3.15、3.42; INF-γ단백:(856±45) pg/ml비(1180±58)pg/ml,IL-2단백:(167±12) pg/ml비(225±16) pg/ml,q=3.11、3.14；균P＜0.05].1.25 mmol/L Z-VAD-FMK조소서이인류림파결중T세포내참입적BrdU적평균형광강도명현저우음성대조조(185±15비298±21,q=3.02,P＜0.05),T세포3충표면활화표기CD69、CD25여Ia양성세포적백분솔역균명현저우음성대조조(7.8％±0.7％비10.5％±1.0％,9.8％±0.8％비14.5％±1.1％,31.2％±2.8％비46.5％±3.2％,q =3.16、3.52、3.11,균P＜0.05).결론 재소서ACD격발전외용엄보caspase억제제Z-VAD-FMK가이억제소서피손국부급이인류림파결중T세포적증식여활화,종이억제소서ACD적발생.
Objective To explore the effects of N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK),a broad caspase inhibitor,on the elicitation of murine allergic contact dermatitis (ACD) and examine the effects on T lymphocytes.Methods 1-fluoro-2,4-dinitrobenzene (DNFB) was used to establish the classical murine model of ACD.Different concentrations of Z-VAD-FMK were applied before ear provocation.Several parameters were detected,including ear swelling degree,weight differences and thickness of ear tissue under microscope between 2 ears.Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels ofThl cytokines (INF-γ and IL-2) in ear tissues.Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect their levels of mRNA and the results were shown as "copies relative to one million housekeeping genes".Local lymph node assay (LLNA)was conducted.Bromodeoxyuridine-flow cytometry was used to detect the proliferation of T lymphocytes in local lymph node and flow cytometry to detect the activation of T lymphocytes.Results The right ear swelling degree,weight differences and thickness between two ears in the 1.25 mmol/L Z-VAD-FMK group were ( 12.6 ± 1.2) × 10-2 mm,(3.1 ±0.2) mg,and ( 12.1 ± 1.1 ) × 10-2 mm respectively.And they were all significantly lower than those of the negative control group( ( 17.4 ± 1.6) × 10 -2 mm,(4.2 ±0.3)mg,( 16.7 ± 1.5) × 10 -2 mm;q =3.25,2.98,3.12,all P ＜0.05 ).The levels of INF-γ and IL-2 in the ear skin lesions of 1.25 mmol/L Z-VAD-FMK group were (856 ±45) and ( 167 ± 12) pg/ml respectively and they were both significantly lower than those of the negative control group( ( 1 180 ±58) and (225 ± 16)pg/ml;q =3.11,3.14,both P ＜0.05).The mRNA levels of the above two cytokines were 152 ± 12 and 96 ±8 respectively and they were both significantly lower than those of the negative control group (220 ± 15and 156 ± 11 ;q =3.15,3.42,both P ＜0.05).In LLNA,the mean intensity of BrdU in T lymphocytes of 1.25 mmol/L Z-VAD-FMK-treated group was significantly weaker than that of the negative control group ( 185 ± 15 vs 298 ±21,q =3.02,P ＜0.05 ).The percents of activation markers-positive T lymphocytes of the Z-VAD-FMK group were 7.8％ ±0.7％,9.8％ ±0.8％ and 31.2％ ±2.8％ respectively and they were all significantly lower than those of the negative control group ( 10.5％ ± 1.0％,14.5％ ± 1.1％,46.5％ ±3.2％,q =3.16,3.52,3.11,all P ＜0.05).Conclusion Topical use of Z-VAD-FMK prior to ear provocation can suppress the proliferation and activation of T lymphocytes in both skin tissues and local lymph nodes and thus result in the inhibitory effect of allergic contact dermatitis.