EPO干预后人脐带间充质干细胞对人肾小管上皮细胞纤维化及归巢能力的影响

Effects of human umbilical cord mesenchymal stem cells after intervention of EPO on fibrosis and homing ability of human renal tubular epithelial cells

目的探讨促红细胞生成素(EPO)干预的人脐带间充质干细胞(HUMSCs)对人肾小管上皮细胞株(HK-2)纤维化过程的影响。方法取HUMSCs和HK-2细胞,分别进行培养。采用Transwell实验和MTT法筛选EPO干预HUMSCs迁移和增殖的最佳浓度为100 IU/mL,采用RT-PCR法和Westernblotting法筛选转化生长因子β1(TGF-β1)诱导HK-2细胞纤维化的最佳浓度为10μg/L。采用Transwell体系进行细胞共培养,将HK-2细胞分为4组,对照组:上室内为单纯培养基,下室内为HK-2细胞和培养基;TGF-β1组:上室内为单纯培养基,下室内为HK-2细胞加入含10μg/L TGF-β1的培养基;HUMSCs干预组:上室内为HUMSCs和培养基,下室内为HK-2细胞加入含10μg/L TGF-β1的培养基;EPO+HUMSCs干预组:上室内为HUMSCs加入含100 IU/mLEPO的培养基,下室内为HK-2细胞加入含10μg/L TGF-β1的培养基。培养72 h后,在显微镜下观察细胞的形态和生长情况,并计数穿膜细胞数。采用RT-PCR法检测HK-2细胞中纤维化因子TGF-β1、纤维连接蛋白(FN)、Ⅳ型胶原(ColⅣ)、α平滑肌肌动蛋白(α-SMA)及归巢因子基质细胞衍生因子(SDF-1)及其受体CXCR4、干细胞因子(SCF)及其受体c-KitmRNA的相对表达量,Westernblotting法测定上述纤维化因子和归巢因子蛋白的相对表达量。结果与对照组比较,TGF-β1组HK-2细胞失去了上皮细胞的形态,形成了具有迁移能力的上皮样细胞和肌成纤维细胞。与对照组比较,TGF-β1组、HUMSCs干预组、EPO+HUMSCs干预组细胞迁移数量均增加,EPO+HUMSCs干预组细胞迁移数量较HUMSCs干预组和TGF-β1组减少(P均<0.01)。与对照组比较,TGF-β1组细胞TGF-β1、FN、ColⅣ、α-SMA、CXCR4、SDF-1、c-Kit、SCFmRNA及蛋白表达水平最高,HUMSCs干预组次之,EPO+HUMSCs干预组最低(P<0.05或<0.01)。结论EPO可促进HUMSCs的增殖和迁移,将HUMSCs定向迁移至受损的HK-2,抑制HK-2分化成具有迁徙能力的无功能的上皮样细胞,从而延缓肾小管上皮细胞的纤维化进展。

Objective To investigate the effects of erythropoietin(EPO)intervening in human umbilical cord mes⁃enchymal stem cells(HUMSCs)on the fibrosis of human renal tubular epithelial cell line HK-2.Methods HUMSCs and HK-2 cells were cultured,respectively.The optimal concentration of EPO in HUMSCs migration and proliferation was 100 IU/mL by Transwell assay and MTT assay,and the optimal concentration of transforming growth factor-β1(TGF-β1)in HK-2 cell fibrosis was 10μg/L by RT-PCR and Western blotting.The HK-2 cells were divided into 4 groups:control group(simple medium was in the upper chamber,and HK-2 cells and medium were in the lower chamber),TGF-β1 group(simple medium was in the upper chamber,and HK-2 cells added with 10μg/L TGF-β1 were in the lower chamber),HUMSCs intervention group(HUMSCs and medium were in the upper chamber,and HK-2 cells added with 10μg/L TGF-β1 medium were in the lower chamber),and EPO+HUMSCs intervention group(HUMSCs added with 100 IU/mL EPO were in the upper chamber,and HK-2 cells added with 10μg/L TGF-β1 were in the lower chamber).After 72 h of cul⁃ture,the morphology and growth of cells were observed under the microscope,and the number of transmembrane cells was counted.RT-PCR was used to detect the relative mRNA expression levels of fibrosis factors TGF-β1,fibronectin(FN),ColⅣ,α-smooth muscle actin(α-SMA),and homing factors stromal cell-derived factor1(SDF-1)and its receptor CXCR4,stem cell factor(SCF)and its receptor c-Kit in the HK-2 cells;the relative protein expression levels of fibrosis factors and homing factors were determined by Western blotting.Results Compared with the control group,after TGF-β1 inter⁃vention,HK-2 cells lost their original epithelial cell morphology and formed epithelioid cells and myofibroblasts with migra⁃tion ability in the TGF-β1 group.Compared with the control group,the number of cell migration increased in the TGF-β1 group,HUMSCs intervention group,and EPO+HUMSCs intervention group;the number of cell migration in the EPO+HUMSCs intervention group was lower than those in the HUMSCs intervention group and TGF-β1 group(all P<0.01).Compared with the control group,the mRNA and protein levels of TGF-β1,FN,ColⅣ,α-SMA,CXCR4,SDF-1,c-Kit and SCF in the TGF-β1 group were the highest,followed by those in the HUMSCs intervention group,and those in the EPO+HUMSCs intervention group were the lowest(P<0.05 or P<0.01).Conclusion EPO can promote the prolifera⁃tion and migration of HUMSCs,direct the migration of HUMSCs to the damaged HK-2,inhibit the differentiation of HK-2 into non-functional epithelial cells with migration ability,and delay the progression of fibrosis of renal tubular epithelial cells.