CAJ | 학술논문

目的观察长效可溶性I型TNF受体(TNFR p55,sTNFR:IgG-Fc)蛋白对免疫诱导肝硬化大鼠药物致肝功能衰竭的保护作用.方法人血清白蛋白(HAS)与完全弗氏佐剂乳化后皮下注射大鼠,使其免疫致敏,末次免疫后10 d采血,检测抗人白蛋白抗体阳性后,每周2次尾静脉注射HSA,6周后形成免疫诱导型肝硬化.成模大鼠分3组,每组15只.模型对照组在肝硬化基础上予D-GalN和脂多糖腹腔注射急性攻击,预处理组在急性攻击前18 h予长效可溶性TNFR p55蛋白腹腔注射,另设0.9％氯化钠溶液正常对照组.观察各组大鼠的一般情况、存活率、肝功能、病理学改变,ELISA法检测血IL-6、IL-22和肝组织中IL-6水平.分光光度法检测肝细胞裂解液中Caspase 3活性.实时PCR检测增殖细胞核抗原(PCNA)、bcl-2、bax和IL-22受体mRNA表达.组间比较采用方差分析.结果模型对照组大鼠12h后精神萎靡、反应减弱,15只中仅存活3只,预处理组大鼠12h均存活(P=0.029 8),且反应灵敏;两组血ALT分别为(6 533±360)和(105±7)U/L(F=151.60,P＜0.01).模型对照组大鼠肝组织再生结节内发生大块或亚大块坏死,纤维间隔保留;TNFR p55蛋白能减轻肝组织炎性反应坏死程度.模型对照组和预处理组大鼠血清IL-6分别为(842.0±12.9)和(91.9±1.6) pg/mL(F=380.30,P＜0.01),肝组织中分别为(26.2±1.2)和(11.1±0.8) pg/mL(F=176.90,P＜0.01),血清中IL-22分别为(167.0±27.8)和(988.0±109.6) pg/mL(F=37.91,P＜0.01).TNFR p55蛋白预处理可使Caspase 3活性减少近60％(F=303.70,P＜0.01)bax表达减少22％(F=108.80,P＜0.01),而bcl-2和PCNA表达分别增加3.6倍和23.0倍(F=115.60,P＜0.01;F=594.20,P＜0.01).结论长效可溶性TNFR p55蛋白可明显改善免疫诱导肝硬化大鼠药物致肝功能衰竭的存活率、肝功能和炎性反应,显示其潜在的临床应用价值.
목적 관찰장효가용성I형TNF수체(TNFR p55,sTNFR:IgG-Fc)단백대면역유도간경화대서약물치간공능쇠갈적보호작용.방법 인혈청백단백(HAS)여완전불씨좌제유화후피하주사대서,사기면역치민,말차면역후10 d채혈,검측항인백단백항체양성후,매주2차미정맥주사HSA,6주후형성면역유도형간경화.성모대서분3조,매조15지.모형대조조재간경화기출상여D-GalN화지다당복강주사급성공격,예처리조재급성공격전18 h여장효가용성TNFR p55단백복강주사,령설0.9％록화납용액정상대조조.관찰각조대서적일반정황、존활솔、간공능、병이학개변,ELISA법검측혈IL-6、IL-22화간조직중IL-6수평.분광광도법검측간세포렬해액중Caspase 3활성.실시PCR검측증식세포핵항원(PCNA)、bcl-2、bax화IL-22수체mRNA표체.조간비교채용방차분석.결과 모형대조조대서12h후정신위미、반응감약,15지중부존활3지,예처리조대서12h균존활(P=0.029 8),차반응령민;량조혈ALT분별위(6 533±360)화(105±7)U/L(F=151.60,P＜0.01).모형대조조대서간조직재생결절내발생대괴혹아대괴배사,섬유간격보류;TNFR p55단백능감경간조직염성반응배사정도.모형대조조화예처리조대서혈청IL-6분별위(842.0±12.9)화(91.9±1.6) pg/mL(F=380.30,P＜0.01),간조직중분별위(26.2±1.2)화(11.1±0.8) pg/mL(F=176.90,P＜0.01),혈청중IL-22분별위(167.0±27.8)화(988.0±109.6) pg/mL(F=37.91,P＜0.01).TNFR p55단백예처리가사Caspase 3활성감소근60％(F=303.70,P＜0.01)bax표체감소22％(F=108.80,P＜0.01),이bcl-2화PCNA표체분별증가3.6배화23.0배(F=115.60,P＜0.01;F=594.20,P＜0.01).결론 장효가용성TNFR p55단백가명현개선면역유도간경화대서약물치간공능쇠갈적존활솔、간공능화염성반응,현시기잠재적림상응용개치.
Objective To investigate whether a novel long-acting tumor necrotic factor (TNF) antagonist (soluble TNF receptor:IgG Fc [sTNFR:IgG-Fc]) can protect hepatocyte damage against liver failure caused by drugs in immunity-induced cirrhotic rats.Methods Wistar rats were repeatedly sensitized by human serum albumin (HSA) emulsified in complete freud adjuvant.The blood was collected at day 10 after the final sensitization.If anti-albumin antibody was positive,the rats were intravenously injected with HSA twice a week.After six weeks,liver cirrhosis was induced by immunity.All the model rats were divided into three groups with 15 each.Liver failure was induced with D-galactosamine/ lipopolysaccharide (LPS) intraperitoneal injection in the rats with liver cirrhosis in model group.The rats in pretreatment group were intraperitoneally injected with long-acting soluble TNF receptor p55 18 h before D-galactosamine/LPS injection.The control group were injected with 0.9％ sodium chloride.General condition,survival rate,liver function and pathological changes were all examined.Serum levels of interleukin (IL)-6,IL-22 and intrahepatic level of IL-6 were detected by enzyme linked immunosorbent assay (ELISA).The activity of Caspase 3 in hepatocyte lysis solution was measured by spectrophotography.Real-time polymerase chain reaction (PCR) was used to detect mRNA expressions of proliferating cell nuclear antigen (PCNA),bcl-2,bax and IL-22 receptor.Data were analyzed by variance analysis among groups.Results Rats in model group were dispirited with poor response after 12 hours and only 3 survived,compared with soluble TNF receptor p55 pre-treated group rats,in which all survived (P=0.029 8) with flexible response.Serum alanine aminotransferase levels in these two groups were (6 533± 360) and (105 ± 7) U/L,respectively.Hepatic regenerative nodule developed massive or submassive necrosis with septal fibrosis in model group,whereas soluble TNF receptor p55 alleviated the inflammatory and necrosis reaction of hepatic tissue.Serum IL-6 levels in model group and pretreatment group were (842.0±12.9) and (91.9±1.6) pg/mL,respectively (F=380.30,P＜0.01).Intrahepatic levels of IL-6 in these two groups were (26.2±1.2) and (11.1±0.8) pg/mL,respectively (F=176.90,P＜0.01),and serum IL-22 levels were (167.0±27.8) and (988.0±109.6) pg/mL,respectively (F=37.91,P＜0.01).Hepatic Caspase-3 activity was reduced by almost 60％ by soluble TNF receptor p55 pretreatment (F=303.70,P＜0.01) and bax expression reduced by 22％ (F=108.80,P＜0.01),while bcl-2 and PCNA expressions were up-regulated by 3.6-folds and 23.0-folds,respectively (F=115.60,P＜0.01; F=594.20,P＜0.01).Conclusions Long acting soluble TNF receptor p55 could improve survival rate,liver function and reduce inflammatory reaction of rats with liver failure induced by drugs on the basis of liver cirrhosis caused by immunity,which indicates that this drug may process a potential therapeutic value.