骨髓间充质干细胞移植对急性肝功能衰竭大鼠高迁移率族蛋白B1表达的影响

Effect of bone marrow mesenchymal stem cell transplantation on expression of high mobility group box 1 protein in rats with acute liver failure

目的探讨采用骨髓间充质干细胞（BMSC）移植对急性肝功能衰竭（ALF）大鼠模型的血清和肝脏组织高迁移率族蛋白B1（HMGB1）表达的影响。方法将90只健康雌性SD实验大鼠按照随机表法分为空白对照组、ALF组以及BMSC移植组,其中ALF组和BMSC移植组的ALF大鼠模型采用900 mg/kg D-Gal N＋10μg/kg脂多糖腹腔注射建立;BMSC移植组在900 mg/kg D-Gal N＋10μg/kg脂多糖腹腔注射后2 h,予以尾静脉注射BMSC悬液1 ml（1.0×10^7/ml）。三组大鼠均在12、24、72 h时间点取血液及肝组织标本,检测大鼠血清ALT和AST,ELISA法检测血清HMGB1表达量,采用苏木素-伊红染色法观察肝组织病理变化,对肝组织HMGB1的表达水平应用免疫组化法进行检测,RTPCR法检测肝组织HMGB1 m RNA。三组大鼠血清生化指标、血清HMGB1浓度以及肝组织HMGB1 m RNA、HMGB1蛋白相对表达量的不同组别之间的比较采用随机区组设计的方差分析,不同组别的大鼠24 h存活率采用秩转换后的方差分析。结果采用900 mg/kg D-Gal N＋10μg/kg脂多糖腹腔注射的方法可成功诱导ALF模型。ALF组大鼠血清ALT、AST水平随病程逐渐升高,在移植后72 h,BMSCs移植组的血清ALT水平明显低于ALF组[（163.5±31.6）vs（639.0±69.8）U/L,P〈0.01],血清AST水平与ALF组相比差别也有统计学意义[（172.8±55.7）vs（724.3±48.8）U/L,P〈0.01]。空白对照组血清HMGB1浓度、肝组织HMGB1 m RNA表达以及肝组织HMGB1蛋白表达在各时间点均较低,ALF组及BMSC移植组上述指标造模成功后均在较高水平,ALF组随时间延长而升高,移植组在BMSC移植后随时间延长逐渐下降。BMSC移植组在移植后24、72 h与ALF组相比血清HMGB1浓度差异具有统计学意义[（146.8±35.2）vs（290.4±30.9）,（90.6±17.4）vs（486.5±57.2）ng/ml,P均〈0.01];肝组织HMGB1 m RNA表达差异具有统计学意义[（0.8±0.2）vs（1.2±0.3）,（0.6±0.2）vs（1.4±0.1）,P均〈0.01];肝组织HMGB1蛋白表达差异具有统计学意义[（3.8±2.2）vs（6.2±1.6）,（2.6±0.8）vs（8.5±3.0）,P均〈0.01]。移植后24 h,空白对照组、ALF组、BMSC移植组大鼠死亡率组间比较差异有统计学意义（χ^2=21.098,P〈0.01）。结论 BMSC移植可以改善ALF大鼠的肝功能以及肝脏病理,同时降低ALF大鼠血清及肝组织中HMGB1的表达。

Objective To investigate the effect of bone marrow mesenchymal stem cell（BMSC） transplantation on expression of high mobility group box 1 protein（HMGB1） in rats with acute liver failure. Methods SD rats were randomly divided into three groups, including control group, ALF group and BMSC transplantation group. After ALF induction the blood and liver tissue samples were collected at 12 h, 24 h and 72 h. Serum ALT and AST levels were measured for each group. Liver pathological changes were observed using HE staining. We evaluated serum HMGB1 concentration by ELISA assay, the protein expression level of liver tissue HMGB1 by immunohistochemistry and the level of HMGB1 m RNA by RTPCR. Results The ALF models were successfully induced by D-Gal N（900 mg/kg） and LPS（10 μg/kg） injection. Serum levels of ALT and AST in the ALF group gradually increased with the progression of the disease. Compared with the ALF group, significant improvement of liver function parameters was observed in the transplantation group. After 72 h transplantation, serum ALT level of BMSC transplantation group was significantly lower than that of the ALF group [（163.5 ± 31.6） vs（639.0 ± 69.8） U/L, P〈0.01]; serum AST level of BMSCs transplantation group was also significantly lower than that of the ALF group [（172.8 ± 55.7） vs（724.3 ± 48.8） U/L, P〈0.01]. The serum HMGB1 concentrations, the HMGB1 m RNA expression and the HMGB1 protein expression in liver tissue of control group were low at all time points, and they increased with time in the ALF group. After BMSC transplantation, the serum HMGB1 concentrations, the HMGB1 m RNA expression and the HMGB1 protein expression decreased with time. The differences of serum HMGB1 concentrations between ALF group and BMSC transplantation group at 24 h and 72 h after transplantation were statistically significant [（146.8 ± 35.2） vs（290.4 ± 30.9）, and（90.6 ± 17.4） vs（486.5 ± 57.2） ng/ml respectively, both P〈0.01]. The differences of HMGB1 m RNA expressions between ALF group and BMSC transplantation group at 24 h and 72 h after transplantation were statistically significant [（0.8 ± 0.2） vs（1.2 ± 0.3）, and（0.6 ± 0.2） vs（1.4 ± 0.1） respectively, both P〈0.01]. The differences of HMGB1 protein expressions between ALF group and BMSC transplantation group at 24 h and 72 h after transplantation were statistically significant [（3.8 ± 2.2） vs（6.2 ± 1.6）,and（2.6 ± 0.8） vs（8.5 ± 3.0） respectively, both P〈0.01]. After 24 h transplantation, the mortality rates among three groups were statistically significant. Conclusion BMSC transplantation can improve the liver function and alleviate pathological change in rats with acute liver failure, and decrease the expression of HMGB1.