间充质干细胞培养上清抑制星状细胞活化治疗肝纤维化的研究

Mesenchymal stem cells-conditioned medium reduces liver fibrosis by regulating hepatic stellate cells

目的:研究骨髓来源间充质干细胞(mesenchymal stem cells,MSC)条件培养基(mesenchymal stem cell-conditioned medium,MSC-CM)对肝纤维化是否有治疗作用及其作用机制。方法 :6周龄SD大鼠随机分为3组(n=8):模型组,治疗组,正常对照组。建立四氯化碳(CCL4)诱导大鼠肝纤维化模型,腹腔注射CCL4,1.5 m L/kg,每周2次,持续8周。MSC培养至3~5代用于MSC-CM的制备,获取的MSC-CM并超滤浓缩约25倍,过滤除菌。在实验第5周起,治疗组经尾静脉注射2 mg/kg MSC-CM,每天1次至第8周末,同时模型组和正常组注射相同剂量的L-DMEM。至实验终点,按照原位灌注和密度梯度离心法提取肝星状细胞(hepatic stellate cells,HSC),并留取肝组织行病理学检查。通过Masson染色和免疫组织化学染色法来检测胶原纤维沉积的程度和α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的表达程度;qRT-PCR和Western blot检测HSCs中α-SMA、转化生长因子β1(transforming growth factorβ1,TGF-β1)、Ⅰ型胶原蛋白(CollagenⅠ)、基质金属蛋白酶-2(matrix metalloproteinases-2,MMP-2)、组织金属蛋白酶抑制剂-2(tissue inhibitor of metalloproteinases-2,TIMP-2)mRNA和蛋白的表达水平。结果:MSC-CM治疗组肝脏炎症程度较模型组减轻,胶原纤维和α-SMA表达量均较模型组明显降低(P<0.05),但未达到正常组水平(P<0.05)。治疗组HSC中TGF-β1、α-SMA、CollagenⅠmRNA和蛋白表达水平较模型组明显下降(P<0.05),MMP-2 mRNA表达水平较模型组升高(P<0.05)。结论:静脉注射MSC-CM对CCL4诱导的肝纤维化有一定治疗作用,这一过程可能与MSC-CM能降低TGF-β1的表达、抑制HSC活化及促进胶原蛋白降解有关。

Objective： To investigate whether the MSC-conditioned medium （MSC-CM） can protect injured liver and reduce liver fibrosis and its potential mechanisms.Methods： Six-week-old SD rats were allocated into three groups （each group n =8） as follows：model group; treatment group; normal group.The liver fibrosis model was established by intraperitoneal injection of low dose of CCL4 （1.5 m L/kg）twice a week for eight weeks.MSCs were grown for 3~5 generation for the preparation of MSC-CM,which was concentrated 25-fold using ultrafiltration.From weeks 5 to 8,MSC-CM was injected every day with a dose of 2 mg/kg by tail vein in the treatment group; at the same time,the other two groups were injected with the same dose of L-DMEM.At the end of the experiment,hepatic stellate cells （HSCs） were isolated by in situ perfusion and density gradient centrifugation,and liver tissue was collected for pathologic analysis.The collagenous fiber was detected by Masson staining,while the expression of alpha-smooth muscle actin （α-SMA） in liver tissues was measured by immunohistochemical staining.To evaluate liver fibrosis,the gene and protein expression ofα-SMA,transforming growth factor beta 1 （TGF-β1）,collagen I,matrix metalloproteinases-2 （MMP-2）,tissue inhibitor of metalloproteinases-2 （TIMP-2） in HSCs were evaluated by quantitative real-time PCR and Western blot.Results： In liver tissues,histological improvement was observed in hepatic fibrosis after MSC-CM treatment （P〈0.01）; the expression of α-SMA and collagenous fiber was significantly lower in the treatment group compared to the model group （P〈0.05）,but not equal to the normal group.In HSC study,the mRNA expressions of TGF-β1,α-SMA,collagen Ⅰin the treatment group were significantly decreased than those in the model group （P〈0.05）; and their protein expressions were also lower in the treatment group （P〈0.05）,and the mRNA expression of MMP-2 increased compared to the model group.Conclusion： Our results showed that MSC-CM has a therapeutic effect on CCL4-induced liver fibrosis.And this process may be related to down-regulaing expression of TGF-β1,inhibiting HSCs activation and promoting collagen degradation.