【摘要】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Westernblot 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hUC-MSC-CM 可能通过抑制Wnt/β-catenin 信号通路，缓解HG诱导HPMCs的EMT。
【Abstract】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-cad-herin: 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..
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