摘要
目的了解微小RNA（miRNA）在急性肝功能衰竭小鼠模型中的表达谱及对其发病机制的调控作用。方法将BALB／c小鼠85只分为4组，模型组40只用D-氨基半乳糖（D-GalN）联合脂多糖（LPS）腹腔注射建立肝功能衰竭模型，D-GalN单药组20只、LPS单药组20只和对照组5只分别予D-GalN、LPS和0．9％NaCl溶液腹腔注射。在给药后0、5和7h观察小鼠肝脏组织学变化，0、1、3、5、7和9h留取小鼠血清和肝脏组织标本。采用实时定量PCR方法检测小鼠血清及肝组织中炎性细胞因子（TNF-α和IL一6）表达水平；采用mIRNA微阵列（microarray）检测小鼠肝脏中miRNA的表达谱，实时定量PCR验证其表达。体外LPS诱导小鼠巨噬细胞Raw264．7活化，诱导不同时间点收集细胞检测细胞miRNA的表达情况。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miRNA微阵列发现小鼠急性肝功能衰竭发病过程中miRNA的表达谱发生了明显变化，与对照组相比，模型组有97个miRNA表达变化显著（P〈0．01），其中21个miRNA在给药后5h和7h均表达上调，27个miRNA均表达下调，进一步PCR验证得到miR-146a和miR-155随给药时间延长表现为持续性上调，且miR-155表达与TNF—α和IL-6表达均具有良好的相关性。体外实验进一步发现miR-146a和miR-155在活化的巨噬细胞中表达上调。结论在小鼠急性肝功能衰竭发病过程中，伴随着miRNA表达谱的变化，炎性反应相关miR-146a和miR-155明显上调，可能在急性肝功能衰竭的免疫发病机制中发挥重要的调控作用。
Objective To study the expression profile of microRNA （miRNA） and the roles in pathogenesis of acute liver failure in mouse model. Methods Eighty-five BALB/e mice were divided into four groups： 40 in model group of acute liver failure were intraperitoneally injected with D- galaetosamine （D-GaIN） and lipopolysaccharides （LPS）; 20 in D-GalN group were injected with D- GalN only; 20 in LPS group were injected with LPS only; 5 in control group were injected with saline. Liver histology of mouse was observed at hour 0, 5, 7 of injection, and sera and liver tissues were collected at hour 0, 1, 3, 5, 7, 9 of injection. Meanwhile, levels of inflammatory factors [-tumor necrosis faetor-α （TNF-α） and interleukin-6 （IL-6）] in serum and liver tissue were detected by realtime polymerase chain reaction （PCR）. Lock nucleic acid （LNA）-based miRNA microarray technologywas used to detect the expression profile of hepatic miRNA, and the expression of miRNA was verified by real time quantification-polymerase chain reaction （RT PCR）. Mouse macrophage Raw264.7 cells were induced by LPS in vitro and the expressions of miRNA at different time points were detected. The comparison of means among groups was analyzed using one way ANOVA and the correlation were analyzed bv Pearson and Spearman correlation. Results Microarray analysis found that the expression profile of miRNA during the acute liver failure changed dramatically. There were 97 miRNA in model group changed significantly compared with control group （P〈0.01）, including 21 up-regulated and 27 down-regulated at hour 5 and 7 of injection. Furthermore, the expressions of miR 146a and miR 155 were verified by RT PCR and found they both increased progressively over time after injection. Correlation analysis showed that miR 155 was well correlated with both TNF-α and IL 6 expressions. It was further found that miR-146a and miR-155 were both up-regulated in activated Raw264.7 cells in vitro. Conclusions The expression profile of miRNA changes
出处
《中华传染病杂志》
CAS
CSCD
北大核心
2010年第12期705-711,共7页
Chinese Journal of Infectious Diseases
基金
基金项目：国家科技部十一五重大传染病专项资助项目（2008ZX10002-005、2008ZX10002-007）
国家自然科学基金资助项目（30872252）
上海市科委面上项目（10411966800）
