人脐带间充质干细胞条件培养液对高糖诱导人腹膜间皮细胞-间充质转化的影响和机制

The effect of conditioned medium from human umbilical cord mesenchymal stem cells on high glucoseinduced epithelial-mesenchymal transition(EMT) in human peritoneal mesothelial cells and its underlying mechanism

目的研究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs)条件培养液(conditioned medium,CM)对高糖(high glucose,HG)诱导人腹膜间皮细胞(human peritoneal mesothelial cells,HPMCs)发生上皮细胞-间充质转化(epithelial-mesenchymal transition,EMT)的影响及其作用机制。方法应用CCK-8法检测不同浓度葡萄糖(1.5%,2.5%,4.25%)和不同体积分数(5%,7.5%,10%,12.5%,15%)的间充质干细胞条件培养液(mesenchymal stem cells conditioned medium,MSC-CM)对HPMCs增殖的影响。实验分组:①对照组;②HG组(2.5%葡萄糖);③CM组(2.5%葡萄糖+7.5%MSC-CM),作用48h。光镜下观察HG和MSC-CM处理后HPMCs的形态学变化。应用Western blot检测HG和MSC-CM处理后HPMCs中EMT标志物以及Wnt/β-catenin通路蛋白的表达。结果 HG抑制HPMCs的增殖,MSC-CM促进HPMCs的增殖。光镜下,HG组细胞形态多数呈梭形改变,MSC-CM可以缓解HG引起的细胞形态学改变。与对照组相比,HG组中E-cadherin表达减少(t=7.166,P=0.002),Vimentin和α-SMA表达升高(t值分别为3.748,3.430;P值分别为0.020,0.027),CM组可缓解HG引起的上述蛋白表达变化(t值分别为3.865,4.657,6.000;P值分别为0.018,0.010,0.004)。与对照组相比,HG组上调Wnt/β-catenin通路中β-catenin蛋白的表达(t=3.341,P=0.029),CM组抑制HG诱导的β-catenin蛋白表达上调(t=3.808,P=0.019)。结论 h UC-MSC-CM可能通过抑制Wnt/β-catenin信号通路,缓解HG诱导HPMCs的EMT。

Objective To explore the effect of conditioned medium(CM) from human umbilical cord mesenchymal stem cells(hUC-MSCs) on high glucose(HG)-induced epithelial-mesenchymal transition(EMT) in human peritoneal mesothelium cells(HPMCs) and its underlying mechanism. Methods The effect of HG and mesenchymal stem cells conditioned medium(MSC-CM) on HPMCs proliferation was detected by CCK-8. HPMCs were divided into three groups: control group, HG group and CM group. Morphology changes of HPMCs were observed under microscope. The expressions of E-cadherin, vimentin, α-SMA and β-catenin in HPMCs was detected by western blot. Results HG reduced the proliferation of HPMCs, and MSC-CM promoted the proliferation of HPMCs. Compared to control group, most HPMCs in HG group became spindle shape, and MSC-CM could alleviate the morphological changes of HPMCs. Compared to control group, HG treatment reduced the expression level of E-cadherin(0.730±0.049 vs. 0.934±0.008, t=7.166,P=0.002), and increased the expression levels of vimentin and α-SMA(vimentin: 0.897 ± 0.037 vs. 0.645 ±0.110, t=3.748, P=0.020;α-SMA: 1.120±0.083 vs. 0.829±0.122, t=3.430, P=0.027), while MSC-CM significantly increased the expression of E-cadherin, and decreased the expressions of vimentin and α-SMA(E-cadherin: 0.894 ± 0.055 vs. 0.730 ± 0.049, t=3.865, P=0.018;vimentin: 0.737 ± 0.046 vs. 0.897 ± 0.037, t=4.657,P=0.010;α-SMA: 0.808±0.036 vs. 1.120±0.083, t=6.000, P=0.004). Meanwhile, HG increased the expression of β-catenin in HPMCs(0.842±0.059 vs. 0.664±0.071, t=3.341, P=0.029), while MSC-CM reduced the expression of β-catenin compared with HG group(0.613±0.086 vs. 0.842±0.059, t=3.808, P=0.019). Conclusions CM from hUC-MSCs attenuate high glucose-induced EMT by down-regulation of Wnt/β-catenin pathway in HPMCs..