骨髓间充质干细胞联合过表达miR-23b增强治疗EAE小鼠的研究

The research on the enhancement therapeutic effects of the mesenchymal stem cells combined with miR-23b on EAE mice

目的验证过表达小RNA(micro RNA,miR)-23b的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)回输对治疗实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠具有更好的协同治疗效果。方法体外实验将EAE小鼠新鲜外周T淋巴细胞与miR23b-BMSCs(miR23b-BMSC-LN组),未处理BMSCs(BMSC-LN组),或未加入BMSCs的空白对照(Control-LN组)共培养48 h,应用酶联免疫吸附法(enzyme linked immunosorbent assay,ELISA)检测其中白细胞介素(interleukin,IL)-17、转化生长因子-β(transforming growth factor,TGF-β)的表达水平。用CD4 Percp、CD25 PE、IL-17 APC、Foxp3 APC抗体对细胞进行染色,流式细胞仪分析MOG35-55刺激后的CD4^(+)IL-17^(+)Th17细胞和CD4^(+)CD25^(+)Foxp3^(+)Treg细胞的百分比;体内实验应用HE染色分析回输miR23b-BMSCs组(miR23b-BMSC-EAE组),回输未处理BMSCs组(BMSCs-EAE组),假手术组(ctrl-EAE组)的脊髓白质的炎性浸润情况。结果过表达miR-23b可增强BMSCs可抑制Th17细胞的分化能力[(4.34±0.32)%比(8.21±0.16)%,χ^(2)=10.03,P<0.05],并促进调节性T(T regular,Treg)细胞亚群的分化[(1.05±0.27)%比(0.24±0.06)%,χ^(2)=12.67,P<0.05]。在病理表现上,miR23b-BMSCs移植能够通过降低Th17/Treg细胞比率以及抑制炎症细胞浸润血脑屏障,从而减轻脊髓脱髓鞘,延缓EAE病程的进展。结论过表达miR23b-BMSCs回输治疗具有更高效更持久的治疗效果,为BMSCs治疗自身免疫疾病的应用奠定了基础。

Objective To improve the treatment of mice with experimental autoimmune encephalomyelitis experimental autoimmune encephalomyelitis(EAE)with bone marrow mesenchymal stem cells(BMSCs)over-expressing microRNA(micro RNA,miR)-23b provided better synergistic and longer-term therapeutic effects.Methods MiR23b-BMSCs(miR23b-BMSC-LN group),BMSCs(BMSC-LN group),or blank control without BMSCs(Control-LN group)were cultured separately for 48 h before commencing co-cultures with fresh peripheral T lymphocytes from EAE mice.After an additional 48 h,the supernatant was collected and tested for expression levels of interleukin(IL)-17,and transforming growth factor-β(TGF-β)in enzyme-linked immunosorbent assay(ELISA).To determine the percentages of CD4^(+)IL-17+T(Th17)cells and CD4^(+)CD25^(+)Foxp3^(+)Treg cells,flow cytometry analysis was conducted with CD4 Percp,CD25 PE,IL-17 APC,and Foxp3 APC antibodies.Spinal cord sections were stained with hematoxylin and eosin(HE)to detect inflammatory cell infiltrates.Results Over-expression of miR-23b enhanced the ability of BMSCs to inhibit differentiation of Th17 cells and reduced IL-17 secretion[(4.34±0.32)%vs(8.21±0.16)%,χ^(2)=10.03,P<0.05].Compared to traditional BMSCs,the miR-23b over-expressing BMSCs(miR23b-BMSCs)promote the differentiation of regulatory T(Treg)cells[(1.05±0.27)%vs(0.24±0.06)%,χ^(2)=12.67,P<0.05].Pathologically,miR23b-BMSC transplantation delayed EAE progression,apparently by reducing the Th17/Treg cell ratio and inhibiting inflammatory cell infiltration across the blood-brain barrier,and thus slowing spinal cord demyelination.Conclusion The addition of miR-23b to BMSCs transplants had potent immunosuppressive and protective effects that served to inhibit EAE development.These results may lead to better utility of BMSCs as a treatment for autoimmune diseases.