白细胞介素10基因修饰的骨髓间充质干细胞抑制大鼠肝纤维化形成的实验研究

Inhibition of liver fibrosis by IL-IO gene-modified BMSCs in a rat model

目的观察pcDNA3．0-白细胞介素10（IL-10）真核表达质粒电穿孔转染的骨髓间充质干细胞（BMSCs）对肝纤维化的治疗效果。方法分离纯化大鼠BMSCs，并进行4，6-二脒基-2-苯基吲哚标记；将60只雄性Wistar大鼠随机分为A、B、C三组，每组20只。采用皮下注射四氯化碳（CCl4）方法建立肝纤维化模型。A组、B组大鼠在造模的同时分别尾静脉注射pcDNA3．0-IL-10-BMSCs和BMSCs细胞悬液1ml，其细胞密度为1×10^6个／ml，每周2次，共8周；C组大鼠尾静脉注射1ml磷酸盐缓冲液。8周后处死大鼠，取肝组织：冰冻切片，观察标记细胞；Masson染色观察纤维化情况；Westem blot检测IL-10表达量；ELISA法检测肿瘤坏死因子仅（TNFα）的含量；碱水解法检测羟脯氨酸（HYP）含量。使用SPSS13．0统计软件包进行方差检验，各实验组间两两比较采用q检验。结果CCl4诱导8周后，Masson染色显示模型鼠大部分形成中度以上肝纤维化；标记的细胞在A组、B组肝组织中均被观察到；纤维化评分和HYP含量：C组分别为（3．15±0．96）μg／mg和（1．89±1．03）μg／mg、A组分别为（1．01±0．35）μg／mg和（0．73±0．29）μg／mg、B组分别为（1．34±0．65）μg／mg和（1．21±0．78）μg／mg，大鼠的肝纤维化程度A组和B组比C组明显减轻，A组比B组程度轻，q值分别为-10．02、-5．01、8．21、4．821、4．73，P值均〈0．01，差异有统计学意义；TNF-α含量在A组为（275．21±86．35）pg／mg、B组为（321．76±98．49）pg／mg、C组为（476．23±126．43）pg／mg，A组和B组均低于C组，A组低于B组，q值分别为-12．86、-8．96和-7．43，．P值均〈0．01，差异有统计学意义。结论应用pcDNA3．0-IL-10-BMSCs能有效抑制CCl4诱导的大鼠肝纤维。

Objective To investigate the potential curative effects of bone marrow mesenchymal stem cells （BMSCs） overexpressing IL-10 for treating liver fibrosis by using a CCl4-induced rat model. Methods BMSCs were cultured and marked by DAPI staining of the cells＇ nuclei. Meanwhile, 60 Wistar rats were treated with CCl4 to induce liver fibrosis and randomly divided into three groups： A： injected with DAPI-marked BMSCs transfected with pcDNA3.0-IL-10; B： injected with DAPI-marked BMSCs; C： injected with phosphate-buffered saline. Histological analysis of excised livers was conducted to observe BMSCs （by DAPI marker） and fibrosis （by Masson＇s stain）. Western blotting was used to detect IL-10 expression in BMSCs. Effects on expression of tumor necrosis factor-alpha （TNFα） were analyzed by enzyme-linked immunosorbent assay, and on hydroxyproline （HYP） levels were analyzed by the acid hydrolysis method. Results Following CCl4-inducement, rat livers showed the characteristic pathological changes of fibrosis and DAPI-marked cells （BMSCs） were observed. Compared with group C （pulmonary fibrosis score： 3.15±0.96; HYP level： 1.894-1.03 μg/mg）, the extent of liver fibrosis was significantly lower in group A （pulmonary fibrosis score： 1.01 ±0.35; HYP level： 0.734-0.29 μg/mg）and group B （pulmonary fibrosis score： 1.34±0.65; HYP level： 1.21 ±0.78 μg/mg）. The therapeutic effects were significantly greater in group A than group B （q=-4.73, P〈0.05）. TNFα protein expression was significantly lower in group A （275.21 ±86.35） and group B （321.76±98.49） than in group C （476.23± 126.43）. The TNFα expression was significantly lower in group A than group B （q -7.43, P〈 0.05）. Conclusion IL-10 genemodified BMSCs are effective for treating liver fibrosis in a CCl4-induced rat model.