中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2012年
3期
149-153
,共5页
多器官功能障碍综合征%泛素%脾脏%树突细胞%主要组织相容性复合物Ⅱ类分子
多器官功能障礙綜閤徵%汎素%脾髒%樹突細胞%主要組織相容性複閤物Ⅱ類分子
다기관공능장애종합정%범소%비장%수돌세포%주요조직상용성복합물Ⅱ류분자
Multiple organs dysfunction syndrome%Ubiquitin%Spleen%Dendritic cell%Major histocompability complex Ⅱ molecule
目的 探讨多器官功能障碍综合征(MODS)脾脏中泛素(Ub)的表达变化及意义,研究泛素化主要组织相容性复合物Ⅱ(MHCⅡ)对树突细胞(DC)活性及免疫调节作用的影响.方法 210只雄性C57BL/6小鼠按随机数字表法分为正常对照组(30只)和MODS组(180只),通过腹腔注射酵母多糖悬液复制MODS模型,MODS组再随机均分为6、12、24、48 h和5～7d、10 ～ 12 d组.采用免疫组化和免疫荧光法分别检测脾脏中Ub蛋白及CD11c+DC细胞和MHCⅡ类分子Ⅰ-Ab的表达；荧光定量逆转录-聚合酶链反应(RT-PCR)检测脾脏内Ub mRNA表达.结果 ①免疫组化显示:与正常对照组比较,MODS组伤后6h脾脏Ub阳性细胞逐渐增多,24 h达高峰[(16.83±0.38)％比(8.60±0.86)％,P＜0.05],后逐渐下降,至10～12 d Ub阳性细胞已显著低于正常对照组[(4.66±0.34)％比(8.60±0.86)％,P＜ 0.05].②荧光定量RT-PCR显示:与正常对照组比较,MODS组伤后6 h Ub mRNA表达增强,至24 h达高峰(2.17±0.20比1.00±0.00,P＜0.01),随后逐渐下降,至10～ 12d表达已显著低于正常对照组(0.72±0.08比1.00±0.00,P＜0.05).③免疫荧光标记显示:与正常对照组比较,MODS组伤后6 h CD1 1c+DC细胞明显增加,至24 h达高峰[(7.55±0.04)％比(2.08±0.13)％,P＜ 0.05],随后明显减少,至10～12 d已接近正常对照组[(2.28±0.06)％比(2.08±0.13)％,P＞0.05].MODS组伤后6 h Ⅰ-Ab阳性细胞明显增加[(10.90±1.40)％比(5.78±0.47)％,P＜0.01],24 h表达明显下降[(3.32±0.91)％比(5.78±0.47)％,P＜0.05],48 h、5～7d恢复至正常对照组水平后,10～12 d再次显著降低[(2.20±0.97)％比(5.78±0.47)％,p＜0.05].Ub阳性细胞数与Ⅰ-Ab、CD11c均呈显著正相关(n=0.899,r2=0.987,均P＜0.05).结论 Ub可能通过泛素化DC表面的MHCⅡ类分子影响DC成熟和活化,从而影响MODS各时期的免疫应答,为调控免疫应答提供新的认识途径与监测指标.
目的 探討多器官功能障礙綜閤徵(MODS)脾髒中汎素(Ub)的錶達變化及意義,研究汎素化主要組織相容性複閤物Ⅱ(MHCⅡ)對樹突細胞(DC)活性及免疫調節作用的影響.方法 210隻雄性C57BL/6小鼠按隨機數字錶法分為正常對照組(30隻)和MODS組(180隻),通過腹腔註射酵母多糖懸液複製MODS模型,MODS組再隨機均分為6、12、24、48 h和5～7d、10 ～ 12 d組.採用免疫組化和免疫熒光法分彆檢測脾髒中Ub蛋白及CD11c+DC細胞和MHCⅡ類分子Ⅰ-Ab的錶達；熒光定量逆轉錄-聚閤酶鏈反應(RT-PCR)檢測脾髒內Ub mRNA錶達.結果 ①免疫組化顯示:與正常對照組比較,MODS組傷後6h脾髒Ub暘性細胞逐漸增多,24 h達高峰[(16.83±0.38)％比(8.60±0.86)％,P＜0.05],後逐漸下降,至10～12 d Ub暘性細胞已顯著低于正常對照組[(4.66±0.34)％比(8.60±0.86)％,P＜ 0.05].②熒光定量RT-PCR顯示:與正常對照組比較,MODS組傷後6 h Ub mRNA錶達增彊,至24 h達高峰(2.17±0.20比1.00±0.00,P＜0.01),隨後逐漸下降,至10～ 12d錶達已顯著低于正常對照組(0.72±0.08比1.00±0.00,P＜0.05).③免疫熒光標記顯示:與正常對照組比較,MODS組傷後6 h CD1 1c+DC細胞明顯增加,至24 h達高峰[(7.55±0.04)％比(2.08±0.13)％,P＜ 0.05],隨後明顯減少,至10～12 d已接近正常對照組[(2.28±0.06)％比(2.08±0.13)％,P＞0.05].MODS組傷後6 h Ⅰ-Ab暘性細胞明顯增加[(10.90±1.40)％比(5.78±0.47)％,P＜0.01],24 h錶達明顯下降[(3.32±0.91)％比(5.78±0.47)％,P＜0.05],48 h、5～7d恢複至正常對照組水平後,10～12 d再次顯著降低[(2.20±0.97)％比(5.78±0.47)％,p＜0.05].Ub暘性細胞數與Ⅰ-Ab、CD11c均呈顯著正相關(n=0.899,r2=0.987,均P＜0.05).結論 Ub可能通過汎素化DC錶麵的MHCⅡ類分子影響DC成熟和活化,從而影響MODS各時期的免疫應答,為調控免疫應答提供新的認識途徑與鑑測指標.
목적 탐토다기관공능장애종합정(MODS)비장중범소(Ub)적표체변화급의의,연구범소화주요조직상용성복합물Ⅱ(MHCⅡ)대수돌세포(DC)활성급면역조절작용적영향.방법 210지웅성C57BL/6소서안수궤수자표법분위정상대조조(30지)화MODS조(180지),통과복강주사효모다당현액복제MODS모형,MODS조재수궤균분위6、12、24、48 h화5～7d、10 ～ 12 d조.채용면역조화화면역형광법분별검측비장중Ub단백급CD11c+DC세포화MHCⅡ류분자Ⅰ-Ab적표체；형광정량역전록-취합매련반응(RT-PCR)검측비장내Ub mRNA표체.결과 ①면역조화현시:여정상대조조비교,MODS조상후6h비장Ub양성세포축점증다,24 h체고봉[(16.83±0.38)％비(8.60±0.86)％,P＜0.05],후축점하강,지10～12 d Ub양성세포이현저저우정상대조조[(4.66±0.34)％비(8.60±0.86)％,P＜ 0.05].②형광정량RT-PCR현시:여정상대조조비교,MODS조상후6 h Ub mRNA표체증강,지24 h체고봉(2.17±0.20비1.00±0.00,P＜0.01),수후축점하강,지10～ 12d표체이현저저우정상대조조(0.72±0.08비1.00±0.00,P＜0.05).③면역형광표기현시:여정상대조조비교,MODS조상후6 h CD1 1c+DC세포명현증가,지24 h체고봉[(7.55±0.04)％비(2.08±0.13)％,P＜ 0.05],수후명현감소,지10～12 d이접근정상대조조[(2.28±0.06)％비(2.08±0.13)％,P＞0.05].MODS조상후6 h Ⅰ-Ab양성세포명현증가[(10.90±1.40)％비(5.78±0.47)％,P＜0.01],24 h표체명현하강[(3.32±0.91)％비(5.78±0.47)％,P＜0.05],48 h、5～7d회복지정상대조조수평후,10～12 d재차현저강저[(2.20±0.97)％비(5.78±0.47)％,p＜0.05].Ub양성세포수여Ⅰ-Ab、CD11c균정현저정상관(n=0.899,r2=0.987,균P＜0.05).결론 Ub가능통과범소화DC표면적MHCⅡ류분자영향DC성숙화활화,종이영향MODS각시기적면역응답,위조공면역응답제공신적인식도경여감측지표.
Objective To explore the expression change in ubiquitin(Ub)in the spleen and its significance in multiple organ dysfunction syndrome(MODS)in mice,and to study the effects of ubiquitinization of major histocompability complex Ⅱ(MHC Ⅱ)on the activity and immunomodulation function of splenic dendritic cell(DC).Methods Two hundred and ten mice were divided into the normal control group(n=30)and MODS group(n=180)according to the method of random digital table,and MODS model was replicated by intraperitoneal injection of zymosan.The MODS group mice were further divided evenly into 6,12,24,48-hour and 5-7-day and 10-12-day groups.Ub protein and expression of CD11c+DC and MHC Ⅱ molecule Ⅰ-Ab were examined using immunohistochemistry and immunofluorescence methods.Ub mRNA expression in the spleen was measured by real-time quantitative reverse transcription-polymerase chain reaction(RT-PCR).Results ①Immunohistochemistry results showed:the number of Ub positive cells in the spleen increased significantly at 6 hours in MODS group compared with that of the normal control group,and it reached the peak at 24 hours[(16.83 ± 0.38)％ vs.(8.60 ± 0.86)％,P＜0.05]and then decreased gradually.At 10-12 days,the number of Ub positive cells decreased significantly compared with that of the normal control group[(4.66 ± 0.34)％ vs.(8.60 ± 0.86)％,P＜0.05].②RT-PCR results displayed:compared with normal control group,Ub mRNA expre ssion in spleen increased at 6 hours in MODS group,and it reached the peak at 24 hours (2.17 ±0.20 vs.1.00 ±0.00,P＜0.01).Then,it decreased gradually.At 10-12 days,Ub mRNA decreased significantly as compared with that of normal control group(0.72±0.08 vs.1.00±0.00,P＜0.05).(③Immunoinfluorescence results displayed:compared with normal control group,CD11c+DC increased significantly at 6 hours in MODS group and rreached the peak at 24 hours[(7.55 ± 0.04)％ vs.(2.08 ± 0.13)％,P＜0.05],and then it decreased gradually.At 10-12 days,it was close to that of the normal control group[(2.28 ±0.06)％ vs.(2.08 ±0.13)％,P＞0.05].Compared with the normal control group,Ⅰ-Ah positive cells in the spleen was significantly increased at 6 hours in MODS group[(10.90 ± 1.40)％ vs.(5.78 ± 0.47)％,P＜0.01],but it decreased at 24 hours [(3.32 ± 0.91)％ vs.(5.78 ± 0.47)％,P＜0.05].Ⅰ-Ab positive cells were restored to the normal level at 48 hours and 5-7 days,and decreased significantly again at 10-12 days[(2.20±0.97)％ vs.(5.78 ±0.47)％,P＜0.05].The number of Ub positive cells correlated positively to the expression of Ⅰ-Ab and the CD11 c(r1 =0.899,r2 =0.987,both P＜0.05).Conclusions Ub might influence the maturation and activation of DC via ubiquination of the MHC Ⅱ molecule on DC,thereby influencing the immune response at different stages of MODS.The result might provide a new way to recognize immune response and also a new monitoring index for immune response regulation.
