摘要目的 探讨间充质干细胞(MSC)对急性肝功能衰竭(ALF)小鼠肝组织中miRNA-155和TNF表达的影响,以及与MSC疗效间的关系.方法 采用随机数字表法和随机数余数分组法将BALB/c清洁级小鼠96只分为4组,即健康对照组、急性肝功能衰竭组、MSC治疗组和MSC预防组各24只.ALF组予以D-GalN 900 mg/kg+脂多糖10 μg/kg腹腔注射建立模型;MSC治疗组在900 mg/kg D-GalN+ 10 μg/kg脂多糖腹腔注射后2h,予以尾静脉注射MSC 1×106;MSC预防组在900mg/kgD-GalN+10 μg/kg脂多糖腹腔注射前予以尾静脉注射MSC 1×106;健康对照组予以0.9％氯化钠溶液1 mL腹腔注射.给药7h后每组处死小鼠12只,检测小鼠血清肝功能,ELISA法检测TNF水平,实时定量PCR检测肝组织miRNA-155、TNF mRNA.余下每组12只小鼠观察24 h生存率.组间均数比较用单因素方差分析,相关性分析采用Pearson和Spearman相关分析.结果 D GalN/脂多糖诱导7h后,MSC预防和MSC治疗组小鼠ALT水平均较ALF组降低,差异均有统计学意义(q值分别为13.20和4.58,均P＜0.01);MSC预防组和MSC治疗组AST水平均较ALF组降低,差异均有统计学意义(q值分别为18.90和9.83,均P＜0.01).与ALF组相比,MSC预防组和MSC治疗组血清TNF水平均有所降低,差异均有统计学意义(q值分别为14.70和3.95,均P＜0.01).健康对照组、ALF组、MSC治疗组和MSC预防组小鼠肝组织TNF mRNA表达水平分别为1.0±0.3、19.2±2.2、6.6±1.7和4.3±0.9,组间比较差异有统计学意义(F=72.24,P＜0.01).与ALF组相比,MSC预防组和MSC治疗组小鼠肝组织TNF mRNA表达水平均有下调,差异均有统计学意义(q值分别为31.40和21.72,均P＜0.01).与ALF组相比,MSC预防组和MSC治疗组小鼠肝组织miRNA-155水平均有下调,差异均有统计学意义(q值分别为19.40和9.58,均P＜0.01).ALF组小鼠肝组织miRNA 155上调和TNFmRNA诱导呈正相关(r＝0.734,P=0.007).MSC预防组和MSC治疗组miRNA-155和TNF mRNA的部分逆转亦呈正相关(r值分别为0.687和0.590,P值分别为0.014和0.044).给药后24 h,健康对照组、ALF组、MSC治疗组和MSC预防组各死亡0/12、10/12、6/12和3/12只,组间比较差异有统计学意义(x2＝19.078,P＜0.01).结论 在MSC干预小鼠ALF发病过程中,可以部分逆转上调的肝组织miRNA-155和TNF,且存在协同性,提示MSC治疗ALF可能通过对肝组织miRNA-155和TNF的调控发生作用.

abstractsObjective To explore the effects of the mesenchymal stem cells (MSC) on the microRNA 155 (miRNA-155) and tumor necrosis factor (TNF) expressions in liver tissue of mice with acute liver failure (ALF),and to explore the relationship between miRNA 155/TNF and the efficacy of MSC.Methods Using a random number table method,and the remainder grouping method of random numbers.A total of 96 BALB/c mice of clean grade were randomly divided into four groups,namely groups of healthy control (intraperitoneal injection of 0.9 ％ NaCl,1 mI),ALF (intraperitoneal injection of D-galactosamine (D-GalN),900 mg/kg body weight,and lipopolysaccharides,10 mg/kg body weight),MSC treatment (1 × 106 MSC tail intravenously injected 2 h after intraperitoneal injection of D-GalN and lipopolysaccharides) and MSC pretreatment (1)× 106 MSC tail intravenously injected before intraperitoneal injection of D-GalN and lipopolysaccharides).Twelve of the 24 mice in each group were sacrificed 7 h after intraperitoneal administration.Liver function,serum TNF level,miRNA-155 and TNF mRNA of liver tissue were detected subsequently.Survival rate at 24 h was observed in the remaining 12 mice in each group.Mean comparison between groups was conducted with ANOVA,and correlation analysis was performed using Pearson and Spearman correlation analysis.Results Seven hours after D-GalN/lipopolysaccharides induction,alanine aminotransferase levels of MSC treatment and MSC pretreatment were significantly lower when compared with those of ALF (q=13.20 and 4.58,respectively; both P＜0.01) ; aspartate aminotransferase levels decreased in MSC treatment and MSC pretreatment groups compared to ALF group,with significant statistical differences (q=18.90 and 9.83,respectively; both P＜0.01).Compared with ALF,serum levels of TNF decreased in MSC treatment and MSC pretreatment and the difference was statistically significant (q=14.70 and 3.95,respectively; both P＜0.01).The TNF mRNA expressions in liver tissue of the healthy control,ALF,MSC treatment and MSC pretreatment were 1.0±0.3,19.2±2.2,6.6±1.7 and 4.3±0.9,respectively.The difference between groups was statistically significant (F 72.24,P＜ 0.01).The TNF mRNA expressions of MSC treatment and MSC pretreatment were down-regulated in comparison with ALF,which showed statistical difference (q=31.40 and 21.72,respectively; both P＜0.01).Compared with ALF,miRNA 155 in liver tissue of MSC treatment and MSC pretreatment were lowered and the differences were significant (q=19.40 and 9.58,respectively; both P＜0.01).The positive correlation between miRNA 155 and TNF mRNA in liver tissue was confirmed in ALF (r＝0.734,P=0.007),MSC treatment (r=0.590,P=0.044) and MSC pretreatment (r=0.687,P=0.014).24 h after administration,the mortalities of the healthy control,ALF,MSC treatment and MSC pretreatment were 0/12,10/12,6/12 and 3/12,respectively.The difference between groups was statistically significant (x2 =19.078,P ＜ 0.01).Conclusion With MSC intervention in ALF,up regulated miRNA-155 and TNF expressions in liver tissue could be synergetic and partially reversed by MSC,suggesting that MSC treatment for ALF may play an important role via regulating miRNA-155 and TNF.