氧化苦参碱协同骨髓间充质干细胞治疗大鼠肝纤维化的实验研究

目的探讨氧化苦参碱（oxymatrine，OM）协同骨髓间充质干细胞（mesenchymal stem cells，MSCs）对四氯化碳（CCl4）诱导的肝纤维化（ hepatic fibrosis，HF）的影响，并初步探索其机制。方法 50只雄性SD大鼠随机分为5组，即正常对照组、模型组、MSCs组、OM组及OM-MSCs联合治疗组，每组10只。除正常对照组外，其余大鼠均复制CCl4诱导的HF模型。造模成功后，MSCs组尾静脉注射MSCs 5×106个/只，1次/周；OM组肌肉注射OM 50 mg/kg，3次/周； OM-MSCs联合治疗组肌肉注射OM 50mg/kg，3次/周，尾静脉注射MSCs 5×106个/只，1次/周。正常对照组、模型组给予等量的生理盐水处理。共给药8周。8周后检测各组大鼠血清ALT及AST水平，利用HE染色和Masson染色评价肝脏病理损伤及细胞外基质沉积情况。利用酶联免疫吸附法检测大鼠血清中白介素-4（IL-4）和白介素-10（IL-10）的表达。结果（1）与正常对照组比较，模型组大鼠血清中ALT、AST水平明显升高，差异有统计学意义（P<0.05）。与模型组比较，8周末OM组、MSCs组及OM+MSCs联合治疗组血清ALT、AST水平显著下降（P<0.05）；但OM+MSCs联合治疗组的血清ALT、AST水平显著低于OM组及MSCs组（P<0.05）。（2）与模型组比较， OM组、MSCs组及OM+MSCs联合治疗组的肝损伤减轻、细胞外基质沉积面积显著减少（P<0.05），且OM+MSCs联合治疗组较OM组、MSCs组更为显著（P<0.05）。（3）与模型组比较，MSCs组、OM+MSCs联合治疗组血清中IL-4水平显著升高（P<0.05），且后两组比较，后者升高更为显著（P<0.05）；OM治疗组IL-4水平亦升高，但差异无统计学意义（ P>0.05）。（4）OM组、MSCs组及OM+MSCs联合治疗组的血清IL-10的水平显著升高（P<0.05），其中OM+MSCs联合治疗组IL-10的水平升高更为显著（P<0.05）。（5）双光子显微镜显示MSCs注射至大鼠体内后未在肝脏定植，OM也未增加MSCs在肝脏的定植。结论 OM可协同MSCs减轻CCl4诱导的HF，这可能与增加血清中IL-4和IL-10水平有关。

英文摘要:

ObjectiveTo investigate whether oxymatrine （OM） could promote mesenchymal stem cell （MSC） therapy in CCl4-induced hepatic fibrosis （HF） in rats and to initially explore its mechanisms. MethodsTotally 50 male SD rats were randomly divided into five groups，i.e.， the normal control group， the model group， the MSC therapy group， the OM therapy group， and the MSC combined OM therapy group， 10 in each group. Except the normal control group， the HF model was duplicated by CCl4 induction. After successful modeling， rats in the MSC therapy group received 5×106 MSCs by intravenous injection via caudal vein， once a week. Rats in the OM therapy group received 50 mg/kg OM by intramuscular injection， three times a week. Rats in MSC combined OM therapy group received 5×106 MSCs by intravenous injection via caudal vein， once a week and 50 mg/kg OM by intramuscular injection three times a week. Equal volume of normal saline was given to those in the normal control group and the model group. All medication lasted for 8 weeks. Serum levels of ALT and AST were detected 8 weeks later. The hepatic histopathological injury and extracellular matrix deposit were assessed using HE and Masson staining. Expressions of serum interleukin-4 （IL-4） and interleukin-10 （IL-10） were detected using enzyme linked immunosorbent assay （ELISA）. Results（1） Compared with the normal control group， serum levels of ALT and AST significantly increased in the model group （P<0.05）. Compared with the model group， serum levels of ALT and AST significantly decreased in the OM therapy group， the MSC therapy group， and the MSC combined OM therapy group at the end of 8 weeks of treatment （P<0.05）. But serum levels of ALT and AST were significantly lower in the MSC combined OM therapy group than in the OM therapy group and the MSC therapy group （P<0.05）. （2） Compared with the model group， the hepatic injury was significantly lessened and the area of extracelluar matrix deposit was significantly reduced in the OM therapy group， the MSC therapy group， and the MSC combined OM therapy group （P<0.05）. Besides， they wer more significant in the MSC combined OM therapy group （P<0.05）. （3） Compared with the model group， the serum IL-4 level was significantly higher in the MSC therapy group and the MSC combined MO group （P<0.05）. It was higher in the MSC combined MO group （P<0.05）. Although the serum IL-4 level also increased in the OM therapy group， but with no statistical difference （P>0.05）. （4） The serum IL-10 level significantly increased in the OM therapy group， the MSC therapy group， and the MSC combined OM therapy group （P<0.05）， and it was the highest in the MSC combined OM therapy group among the three groups （P<0.05）. （5） Two-photon fluorescence imaging showed no signals of MSCs in liver with or without OM injection. ConclusionOM could promote mesenchymal stem cell therapy in hepatic fibrosis rats， which might be involved in increasing serum levels of IL-4 and IL-10.

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