骨髓间充质干细胞治疗百草枯致大鼠肺纤维化的效果研究

Effect of bone marrow mesenchymal stem cells on paraquat-induced pulmonary fibrosis in rats

目的研究大鼠百草枯(paraquat,PQ)中毒后不同时期输注骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)治疗肺纤维化的疗效并探讨可能的机制。方法于2018年10至12月,采用全骨髓贴壁培养法分离、提纯SPF级SD大鼠的BMSC培养至第三代(P3)。对P3 BMSC进行表面抗原CD29、CD90、CD45、CD34检测并进行成骨和成脂诱导分化培养,分别以碱性磷酸酶(alkaline phosphatase,ALP)、茜素红染色和油红O染色观察ALP、钙结节和脂肪滴形成情况。随机选择36只SPF级SD雄性大鼠随机分成6组,分别是空白对照组(NC组,注射等量的生理盐水),百草枯模型组(PQ组,腹腔注射20%PQ溶液18 mg/kg),BMSC-A组、BMSC-B组、BMSC-C组和BMSC-D组(分别于PQ染毒后3 h、3 d、7 d和14 d注射BMSC悬浮液1×106 cells/只)。28 d后处死,计算各组大鼠的肺脏器系数,碱水解法计算肺组织中羟脯氨酸(hydroxyproline,HYP)含量,HE染色及Masson染色观察肺损伤及肺纤维化情况,ELISA法检测血清中转化生长因子β1(TGF-β1)、肿瘤坏死因子-α(TNF-α)、基质金属蛋白酶-9(MMP-9)及金属蛋白酶组织抑制因子-1(TIMP-1)等细胞因子的水平。结果成功分离并获得高纯度BMSC,P3 BMSC阳性表达CD29、CD90,阴性表达CD34、CD45,并具有成骨、成脂分化潜能。HE染色和Masson染色显示,NC组大鼠肺泡结构完整,均匀;PQ组肺泡结构严重破坏,大量的胶原纤维和成纤维细胞沉积;与PQ组比较,各BMSC组肺损伤程度明显减轻,以BMSC-A组和BMSC-B组减轻程度明显。与NC组比较,PQ组大鼠的肺脏器系数、肺组织中HYP含量及血清中的TGF-β1及TIMP-1水平增高,差异均有统计学意义(P<0.05),而TNF-α、MMP-9差异无统计学意义(P>0.05);与PQ组比较,BMSCA、B组的肺脏器系数、血清中TGF-β1、TIMP-1降低,差异均有统计学意义(P<0.05);与PQ组比较,BMSC-C、BMSC-D组的肺脏各系数血清中TGF-β1、TIMP-1降低,差异无统计学意义(P>0.05)。结论早期注射BMSC对于减缓百草枯导致的肺纤维化效果更好,其机制可能是BMSC通过降低体内TGF-β1水平,调节TIMP-1/MMP-9的平衡,进而减轻炎症损伤、增加细胞外基质的降解而减轻肺纤维化。

Objective To study the effect of the injected bone marrow mesenchymal stem cells(BMSC)on rats with pulmonary fibrosis induced by paraquat(PQ)during different poisoning periods and explore the potential mechanism.Methods From October to December 2018,BMSCs of SPF SD rats were isolated and purified by whole-bone marrow adherent culture method and cultured to the Third Generation(P3).The surface antigens CD29,CD90,CD45 and CD34 of P3 BMSC were detected by Flow cytometry,the formation of alkaline phosphatase(ALP),calcium nodules and fat droplets were observed by ALP,Alizarin Red staining and oil red O staining.At the same time,36 SPF male rats were randomly divided into 6 groups:NC Group(Blank Control Group,injected with the same amount of saline)and PQ group(PQ model group,injected with 20%PQ solution 18 mg/kg intraperitoneally),bMSC-A group,BMSC-B group,BMSC-C group and BMSC-D group were injected with BMSC suspension 1×106 cells/mice at 3 h、3 d、7 d and 14 d after PQ poisoning.After 28 days,the rats were killed,the lung organ coefficients were calculated,the hydroxyproline(HYP)content in lung tissue was calculated by alkaline hydrolysis,and the lung injury and fibrosis were observed by HE and Masson staining,serum TGF-1、TNF-α、MMP-9 and TIMP-1 were detected by Elisa.Results High Purity BMSCs were successfully isolated and obtained.The P3 BMSC generation was positive expression of CD29、CD90、and negative expression of CD34、CD45,and had the potential of osteogenic and adipogenic differentiation.The results of HE staining and Masson staining showed that the alveolar structure in NC group was intact and homogeneous,in PQ group,the alveolar structure was severely damaged and a lot of collagen fibers and fibroblasts were deposited,and the degrees of lung injury in each BMSC intervention group were obviously less than in PQ group,in BMSC-A group and BMSC-B group,the degrees of reduction were obvious.Compared with NC group,the Lung organ coefficient,HYP content in lung tissue and TGF-β1,TIMP-1 levels in serum were significantly higher in PQ group(P<0.05),while TNF-αand MMP-9 had no significant difference(P>0.05).Compared with PQ group,the lung organ Coefficients,HYP,TGF-1 and TIMP-β1 in BMSC-A and BMSC-B groups were lower than those in PQ group(P<0.05).The Lung organ coefficients,TGF-β1 and TIMP-1 in BMSC-C and BMSC-D groups were lower than those in PQ group,there was no significant difference(P>0.05).Conclusion Early BMSC injecting can alleviate pulmonary fibrosis induced by PQ.The mechanism may be that BMSC can reduce pulmonary fibrosis through reducing the level of TGF-β1 and regulating the balance of TIMP-1/MMP-9,threrby reducing inflammatory damage and increasing the degradation of extracellular matrix(ECM).