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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 mesenchymal stem cell-conditioned medium (MSC-CM) can treat liver fibrosis and its mechanism. Methods: Six-week-old SD rats were randomly divided into three groups (n = 8): model group, treatment group and normal control group. A rat model of hepatic fibrosis induced by carbon tetrachloride (CCL4) was established. CCL4 was intraperitoneally injected at 1.5 m L / kg twice a week for 8 weeks. MSC was cultured for 3 to 5 generations for the preparation of MSC-CM. The obtained MSC-CM was concentrated by ultrafiltration for about 25 times and sterilized by filtration. From the fifth week of the experiment, the treatment group was injected with 2 mg / kg MSC-CM through the tail vein once a day until the end of the 8th week. At the same time, the same dose of L-DMEM was injected into the model group and the normal group. At the end of the experiment, hepatic stellate cells (HSC) were extracted by in situ perfusion and density gradient centrifugation, and the liver tissues were collected for histopathological examination. Masson staining and immunohistochemical staining were used to detect the degree of collagen deposition and the expression of α-smooth muscle actin (α-SMA). QRT-PCR and Western blot were used to detect α-SMA , Transforming growth factor β1 (TGF-β1), collagen Ⅰ, matrix metalloproteinases-2 (MMP-2), tissue inhibitor of tissue inhibitor of metalloproteinase- metalloproteinases-2, TIMP-2) mRNA and protein expression levels. Results: Compared with model group, the expression of collagen fibers and α-SMA in MSC-CM group was lower than that in model group (P <0.05), but not in normal group (P <0.05). The expression of TGF-β1, α-SMA, CollagenⅠmRNA and protein in HSC group were significantly lower than those in model group (P <0.05), while the expression of MMP-2 mRNA was higher in model group than in model group (P <0.05). Conclusion: Intravenous injection of MSC-CM has a certain therapeutic effect on CCL4-induced hepatic fibrosis. This process may be related to the fact that MSC-CM can reduce the expression of TGF-β1, inhibit the activation of HSC and promote the degradation of collagen.