中华传染病杂志
中華傳染病雜誌
중화전염병잡지
CHINESE JOURNAL OF INFECTIOUS DISEASES
2015年
6期
351-357
,共7页
董进中%王丽萍%张赛男%施春玮%杨乃彬%倪顺兰%卢明芹
董進中%王麗萍%張賽男%施春瑋%楊迺彬%倪順蘭%盧明芹
동진중%왕려평%장새남%시춘위%양내빈%예순란%로명근
肝功能衰竭,急性%脂多糖类%树突细胞%NF-κB%锌指蛋白 A20
肝功能衰竭,急性%脂多糖類%樹突細胞%NF-κB%鋅指蛋白 A20
간공능쇠갈,급성%지다당류%수돌세포%NF-κB%자지단백 A20
Liver failure,acute%Lipopolysaccharide%Dendritic cells%NF-kappa B%Zinc finger protein A20
目的：观察内毒素耐受 CD11clow CD45RBhigh DC 对急性肝功能衰竭小鼠锌指蛋白 A20表达的影响，探讨其在急性肝功能衰竭治疗中的作用及可能机制。方法建立内毒素耐受小鼠模型，经免疫磁珠法分选正常小鼠脾脏来源 CD11clow CD45RBhigh DC 和内毒素耐受小鼠脾脏来源 CD11clow CD45RBhigh DC。选取健康雄性 BALB／c 小鼠126只，随机分为正常对照组（A 组6只）、急性肝功能衰竭组（B 组40只）、正常小鼠脾脏 CD11clow CD45RBhigh DC 治疗组（C 组40只）和内毒素耐受小鼠脾脏CD11clow CD45RBhigh DC 治疗组（D 组40只）；B、C、D 三组小鼠同时腹腔注射 D-GalN 600 mg／kg和脂多糖10μg／只，A 组注射等体积0．9％氯化钠溶液；30 min 后 C 组腹腔注射正常小鼠脾脏来源 CD11clow CD45RBhigh DC（1×106／只，0．2 mL／只），D 组腹腔注射内毒素耐受小鼠脾脏来源 CD11clow CD45RBhigh DC（1×106／只，0．2 mL／只）进行干预，A、B 组则给予相同剂量的0．9％氯化钠溶液。检测各组小鼠各时间点血 ALT、AST 水平；采用 HE 染色观察各组小鼠各时间点肝组织病理变化；反转录（RT）-PCR 和Western 免疫印迹法检测各组小鼠各时间点 TNF-α、核因子-κB、锌指蛋白 A20表达情况。样本均数比较采用单因素方差分析。进行正态性检验和方差齐性分析，方差齐者采用 LSD 检验。结果 B 组在造模2 h 后 ALT、AST 开始逐渐升高，于24 h 达高峰，明显高于 A 组（t 值分别为31．00和11．52，均 P ＜0．05）。C、D 两组于造模2 h 后 ALT、AST 也逐渐升高，于24 h 达高峰，与 B 组比较，差异均有统计学意义（t 值分别为14．60和26．43，均 P ＜0．05）。B 组小鼠 TNF-αmRNA（t ＝427．58）、核因子-κB mRNA （t＝122．42）及蛋白表达量（t 值分别为179．35和165．98）随着时间的推移逐渐升高，至12 h 达高峰，与A 组相比差异有统计学意义（均 P ＜0．05）。B 组小鼠锌指蛋白 A20 mRNA 及蛋白则随着时间的延长逐渐降低，在12 h 达到最低，与 A 组相比差异有统计学意义（t 值分别为90．80和160．43，均 P ＜0．05）；A20 mRNA 及蛋白表达量则随着时间的延长逐渐增加，在12 h 达到最高值，且 D 组增加较 C 组更明显，差异有统计学意义（t 值分别为11．21和24．80，均 P ＜0．05）。结论内毒素耐受小鼠脾脏来源CD11clow CD45RBhigh DC 治疗可减轻肝脏损害。
目的：觀察內毒素耐受 CD11clow CD45RBhigh DC 對急性肝功能衰竭小鼠鋅指蛋白 A20錶達的影響，探討其在急性肝功能衰竭治療中的作用及可能機製。方法建立內毒素耐受小鼠模型，經免疫磁珠法分選正常小鼠脾髒來源 CD11clow CD45RBhigh DC 和內毒素耐受小鼠脾髒來源 CD11clow CD45RBhigh DC。選取健康雄性 BALB／c 小鼠126隻，隨機分為正常對照組（A 組6隻）、急性肝功能衰竭組（B 組40隻）、正常小鼠脾髒 CD11clow CD45RBhigh DC 治療組（C 組40隻）和內毒素耐受小鼠脾髒CD11clow CD45RBhigh DC 治療組（D 組40隻）；B、C、D 三組小鼠同時腹腔註射 D-GalN 600 mg／kg和脂多糖10μg／隻，A 組註射等體積0．9％氯化鈉溶液；30 min 後 C 組腹腔註射正常小鼠脾髒來源 CD11clow CD45RBhigh DC（1×106／隻，0．2 mL／隻），D 組腹腔註射內毒素耐受小鼠脾髒來源 CD11clow CD45RBhigh DC（1×106／隻，0．2 mL／隻）進行榦預，A、B 組則給予相同劑量的0．9％氯化鈉溶液。檢測各組小鼠各時間點血 ALT、AST 水平；採用 HE 染色觀察各組小鼠各時間點肝組織病理變化；反轉錄（RT）-PCR 和Western 免疫印跡法檢測各組小鼠各時間點 TNF-α、覈因子-κB、鋅指蛋白 A20錶達情況。樣本均數比較採用單因素方差分析。進行正態性檢驗和方差齊性分析，方差齊者採用 LSD 檢驗。結果 B 組在造模2 h 後 ALT、AST 開始逐漸升高，于24 h 達高峰，明顯高于 A 組（t 值分彆為31．00和11．52，均 P ＜0．05）。C、D 兩組于造模2 h 後 ALT、AST 也逐漸升高，于24 h 達高峰，與 B 組比較，差異均有統計學意義（t 值分彆為14．60和26．43，均 P ＜0．05）。B 組小鼠 TNF-αmRNA（t ＝427．58）、覈因子-κB mRNA （t＝122．42）及蛋白錶達量（t 值分彆為179．35和165．98）隨著時間的推移逐漸升高，至12 h 達高峰，與A 組相比差異有統計學意義（均 P ＜0．05）。B 組小鼠鋅指蛋白 A20 mRNA 及蛋白則隨著時間的延長逐漸降低，在12 h 達到最低，與 A 組相比差異有統計學意義（t 值分彆為90．80和160．43，均 P ＜0．05）；A20 mRNA 及蛋白錶達量則隨著時間的延長逐漸增加，在12 h 達到最高值，且 D 組增加較 C 組更明顯，差異有統計學意義（t 值分彆為11．21和24．80，均 P ＜0．05）。結論內毒素耐受小鼠脾髒來源CD11clow CD45RBhigh DC 治療可減輕肝髒損害。
목적：관찰내독소내수 CD11clow CD45RBhigh DC 대급성간공능쇠갈소서자지단백 A20표체적영향，탐토기재급성간공능쇠갈치료중적작용급가능궤제。방법건립내독소내수소서모형，경면역자주법분선정상소서비장래원 CD11clow CD45RBhigh DC 화내독소내수소서비장래원 CD11clow CD45RBhigh DC。선취건강웅성 BALB／c 소서126지，수궤분위정상대조조（A 조6지）、급성간공능쇠갈조（B 조40지）、정상소서비장 CD11clow CD45RBhigh DC 치료조（C 조40지）화내독소내수소서비장CD11clow CD45RBhigh DC 치료조（D 조40지）；B、C、D 삼조소서동시복강주사 D-GalN 600 mg／kg화지다당10μg／지，A 조주사등체적0．9％록화납용액；30 min 후 C 조복강주사정상소서비장래원 CD11clow CD45RBhigh DC（1×106／지，0．2 mL／지），D 조복강주사내독소내수소서비장래원 CD11clow CD45RBhigh DC（1×106／지，0．2 mL／지）진행간예，A、B 조칙급여상동제량적0．9％록화납용액。검측각조소서각시간점혈 ALT、AST 수평；채용 HE 염색관찰각조소서각시간점간조직병리변화；반전록（RT）-PCR 화Western 면역인적법검측각조소서각시간점 TNF-α、핵인자-κB、자지단백 A20표체정황。양본균수비교채용단인소방차분석。진행정태성검험화방차제성분석，방차제자채용 LSD 검험。결과 B 조재조모2 h 후 ALT、AST 개시축점승고，우24 h 체고봉，명현고우 A 조（t 치분별위31．00화11．52，균 P ＜0．05）。C、D 량조우조모2 h 후 ALT、AST 야축점승고，우24 h 체고봉，여 B 조비교，차이균유통계학의의（t 치분별위14．60화26．43，균 P ＜0．05）。B 조소서 TNF-αmRNA（t ＝427．58）、핵인자-κB mRNA （t＝122．42）급단백표체량（t 치분별위179．35화165．98）수착시간적추이축점승고，지12 h 체고봉，여A 조상비차이유통계학의의（균 P ＜0．05）。B 조소서자지단백 A20 mRNA 급단백칙수착시간적연장축점강저，재12 h 체도최저，여 A 조상비차이유통계학의의（t 치분별위90．80화160．43，균 P ＜0．05）；A20 mRNA 급단백표체량칙수착시간적연장축점증가，재12 h 체도최고치，차 D 조증가교 C 조경명현，차이유통계학의의（t 치분별위11．21화24．80，균 P ＜0．05）。결론내독소내수소서비장래원CD11clow CD45RBhigh DC 치료가감경간장손해。
Objective This study aimed to investigate the effect of splenic CD11clow CD45RBhigh dendritic cell (DC)derived from endotoxin tolerance (ET)mice on the expression of zinc finger protein A20 in acute liver failure (ALF)and to clarify the possible mechanism.Methods ET mice were modeled. CD11clow CD45RBhigh DC were isolated from spleen by magnetic activated cell sorting (MACS).One hundred and twenty-six healthy male BALB/c mice were randomly divided into four groups:control group (group A,n=6),ALF group (group B,n =40),normal CD11clow CD45RBhigh DC-treated group (group C,n=40),ET-CD11clow CD45RBhigh DC-treated group (group D,n=40).Mice in group B,C and D were injected with D-galactosamine (D-GalN)600 mg/kg and lipopolysaccharides (LPS)10 μg/mouse.Mice in group A were given the same volume of normal saline (NS).Half an hour after the D-GalN/LPS injection,mice in group C were treated with splenic CD11clow CD45RBhigh DC derived from normal mice (1 ×10 6/mouse,0.2 mL/mouse).Mice in group D were treated with splenic CD11clow CD45RBhigh DC derived from ET mice (1 × 10 6/mouse,0.2 mL/mouse).Mice in group A and B were given the same volume of 0.9% NaCl solution (0.2 mL/mouse).Alanine aminotransferase (ALT)and aspartate aminotransferase (AST)levels were measured at each time point.Liver histopathological changes were confirmed by hematoxglin and eosin methods.Expressions of tumor necrosis factor-α (TNF-α),nuclear factor-kappa B (NF-κB),and zinc finger protein A20 were measured by reverse transcriptase polymerase chain reaction(RT-PCR)and Western blot.One-way analysis of variance was used to compare means between groups.Normal distribution and homogeneity of variance were tested.LSD test was conducted in patients accorded with homogeneity of variance.Results ALT and AST levels increased 2 h after modeling in group B and peaked at 24 h,which were significantly higher than groups A (t = 31 .00, 11 .52,both P <0.05).ALT and AST levels also increased after 2 h after modeling and peaked at 24 h in group C and group D,which were both significantly higher than group B (t =14.60,26.43,both P <0.05).The mRNA levels and protein expressions of TNF-αand NF-κB in group B increased gradually and peaked at 12 h after D-GalN/LPS injection.Compared to that of group A,the differences were both statistically significant (t = 427.58,122.42,179.35 ,165 .98,all P < 0.05 ).The mRNA level and protein expression of zinc finger protein A20 in group B decreased gradually and reached the minimum at 12 h after D-GalN/LPS injection,which was statistically different compared to group A (t = 90.80, 160.43,both P <0.05).On the contrary,the levels of zinc finger protein A20 in group C and D increased gradually and peaked at 12 h after D-GalN/LPS injection.The expression level of zinc finger protein A20 in group D was significantly higher than group C (t = 11 .21 ,24.80,both P < 0.05 ).Conclusion Treatment of splenic CD11clow CD45RBhigh DC derived from ET mice contributes to liver protection against D-GalN/LPS-induced ALF.
