脂肪间充质干细胞对硬皮病小鼠的疗效及其对Wnt/β-catenin信号通路的影响

The therapeutic effects of adipose-derived mesenchymal stem cells on scleroderma mice and its effects on Wnt/β-catenin signaling pathways

目的:探讨脂肪间充质干细胞(ADSCs)对硬皮病(SD)小鼠的治疗效果及其对Wnt/β-catenin信号通路的影响。方法:将40只C57BL/6J雌性小鼠随机平均分为空白组、PBS组、模型组及治疗组。模型组与治疗组通过背部皮下注射博来霉素(BLM)制备SD小鼠模型,空白组不予任何处理,PBS组注射同体积PBS溶液。造模成功后采用100μL ADSCs细胞悬液(2×1010个/L)皮下注射至治疗组小鼠背部局部皮下,每日1次,连续4周。观察各组小鼠皮肤组织病理改变,并检测其皮肤组织中羟脯氨酸含量、脾细胞中辅助性T细胞(Th)17和调节性T细胞(Treg)比例以及血清炎性因子水平。采用western blot检测皮肤组织中Wnt/β-catenin信号通路相关蛋白表达水平。结果:与空白组和PBS组相比,模型组Th17和Treg比例、血清白细胞介素(IL)-6、IL-17、Wnt2、Wnt3a及β-catenin蛋白水平均显著升高(P<0.05),但IL-10水平显著降低(P<0.05)。经ADSCs治疗后SD小鼠皮损组织硬化和炎症均得到明显改善,且羟脯氨酸含量显著降低(P<0.05)。与模型组相比,治疗组Th17比例、血清IL-6及IL-17水平、Wnt2、Wnt3a及β-catenin蛋白表达均降低(P<0.05),而Treg比例及血清IL-10水平均显著升高(P<0.05)。结论:ADSCs能够通过调节T淋巴细胞和相关炎性因子分泌,改善SD小鼠机体炎症反应和皮肤组织纤维化,且该过程可能与Wnt/β-catenin信号通路有关。

Objective: To investigate the therapeutic effects of adipose-derived mesenchymal stem cells(ADSCs) on scleroderma(SD) mice and to explore its effects on Wnt/β-catenin signaling pathways. Methods: Forty C57 BL/6 J female mice were randomly divided into blank group, PBS group, model group and treatment group. The SD model was established by subcutaneous injection of bleomycin(BLM). The blank group received no treatment, and the PBS group was injected with PBS solution. ADSC suspension(concentration 2×1010/L,100 μL) was injected subcutaneously to the back of the mice, once daily for 4 weeks. The pathological changes in the skin, content of hydroxyproline, proportion of Th17 and Treg in the spleen, and the levels of serum inflammatory cytokines were analyzed. Western blot was used to detect the expression levels of Wnt/β-catenin signaling pathway related proteins in the skin. Results: In addition to elevated levels of Wnt2, Wnt3 a and β-catenin proteins in the skin, SD mice also displayed higher levels of serum IL-6 and IL-17, and increased proportion of Th17 and Treg, along with reduction in serum IL-10(P<0.05 vs. blank and PBS controls). Treatments of SD mice with ADSCs significantly improved sclerosis and inflammation, and lowered the content of hydroxyproline significantly(P<0.05). Moreover, ADSC treatments decreased proportion of Th17, serum IL-6 and IL-17 levels while increasing Treg ratio and serum IL-10 levels(P<0.05 vs. untreated SD controls). Finally, ADSC treatments decreased levels of Wnt2, Wnt3 a and β-catenin proteins(P<0.05 vs.untreated SD controls). Conclusion: ADSCs can regulate T lymphocytes and their secretion of cytokines, possibly with the involvement of Wnt/β-catenin signaling pathway, to improve the inflammatory response and fibrosis in SD mice.