青蒿琥酯对人脐带间充质干细胞诱导的去分化肌成纤维细胞凋亡的影响

Artesunate induces apoptosis of human umbilical cord mesenchymal stem cells-induced dedifferentiated myofibroblasts(MSC-deMFB)

目的了解人脐带间充质干细胞(hUCMSC)对肺肌成纤维细胞(MFB)的作用,探索青蒿琥酯联合hUCMSC对肺MFB的作用。方法胶原酶消化贴壁法获得hUCMSC;人胚肺成纤维细胞(MRC-5)经TGF-β1(10 ng/mL)诱导48 h后,加入MFB和(或)青蒿琥酯共培养24 h;撤除MSC后,在MSC-deMFB体系中重新加TGF-β1再刺激。流式检测细胞凋亡、CCK8检测细胞增殖及qPCR和WB分别检测纤维化相关蛋白的mRNA和蛋白表达水平。结果hUCMSC符合ISCT的MSC标准。经TGF-β1刺激后的MRC-5(即MFB)与hUCMSC共培养24 h后,细胞(MSC-deMFB)呈增殖状态,CCK8检测发现OD值较对照组增加(P<0.01);α-SMA、COL1A1及FN1的表达明显降低,接近FB(MRC-5)初始水平(P<0.01)。当撤除MSC后,在MSC-deMFB体系中重新加入TGF-β1时,细胞恢复MFB特征(α-SMA、COL1A1及FN1的表达升高),提示MSC诱导MFB去分化是可逆的。当青蒿琥酯和MSC同时作用MFB时,细胞总凋亡率较对照组增加;并进一步抑制α-SMA、COL1A1的表达(P<0.01)。结论间充质干细胞分泌物诱导肺肌成纤维细胞增殖、可逆性去分化及抑制细胞外基质合成;青蒿琥酯诱导MSC-deMFB凋亡,与MSC协同抑制MSC-deMFB细胞α-SMA及纤维化相关细胞外基质基因表达。青蒿琥酯协同人脐带间充质干细胞可能是抗纤维化的新策略。

Objective To understand the effect of human umbilical cord mesenchymal stem cells(hUCMSCs)on myofibroblasts and to explore the effect of artesunate combined with hUCMSCs on myofibroblasts.Methods hUC-MSCs were isolated from umbilical cord by typeⅡcollagenase digestion and adherence method.Human embryonic lung fibroblasts(MRC-5)were induced by TGF-β1 for 48 h,then added to MSCs and or ART for 24 h.Besides,after removal of the MSCs,TGF-β1 was readded into the MSC-deMFB system.The apoptosis level and the proliferation of cells were detected by flow cytometry and CCK8,respectively.The m RNA and protein expression levels ofα-SMA,collagen and fibronectin were detected by qPCR and WB,respectively.Results hUCMSCs used in the experiment met the ISCT MSC criteria.After TGF-β1 pretreatment of MRC-5(MFB),cells were co-cultured with MSCs for 24 h and the CCK8 increased compared with the control group(P<0.01).The mRNA and protein expression ofα-SMA,COL1A1 and Fibronectin were significantly lower and close to the initial level of fibroblasts.When TGF-β1 was re-added to the MSC-deMFB system,the cells recovered myofibroblasts characteristics,suggesting that MSC-induced myofibroblasts dedifferentiation was reversible.When myofibroblasts were stimulated by artesunate and MSCs simultaneously,the cell apoptosis rate increased compared with the control group and it further inhibited the mRNA and protein expressions ofα-SMA and COL1A1(P<0.01).Conclusions hUCMSCs can induce proliferation,dedifferentiation and inhibit extracellular matrix synthesis of lung myofibroblasts.Artesunate can induce the apoptosis of MSC-deMFB and overcome the effects of MSC-induced proliferation of myofibroblasts.Artesunate in conjunction with hUCMSCs may be a novel anti-fibrosis strategy.