Poly(I:C)影响间充质干细胞的成骨分化及其对树突状细胞的作用

摘要

目的:间充质干细胞(MSC)能够表达Toll样受体(TLR),但TLR配体刺激对MSC的影响依然存在争议.本研究分析了TLR3配体刺激对脐带MSC表型、细胞增殖、凋亡、成骨分化和功能的影响.方法:用混合酶法分离培养了人脐带来源的MSC；用流式细胞仪分析了TLR3配体—Poly(I:C)刺激后MSC表面标志CD29、CD44、CD105、CD31、CDl17和CD45表达的变化；用CCK-8和Annexin-V/PI法分别检测了不同浓度Poly(I:C)刺激24及48小时后MSC的增殖和凋亡情况；进一步分析了Poly(I:C)对MSC成骨分化的影响.此外在Poly(I:C)和MSC存在时,流式检测了DC成熟表面标志CD40、CD86、MHCⅡ的表达,DC对右旋糖酐的吞噬,混合淋巴实验检测了DC的抗原递呈能力.结果:Poly(I:C)的刺激不影响MSC的表型和增殖；只有高浓度组的Poly(I:C)刺激引起MSC的凋亡；Poly(I:C)抑制成骨相关基因RUNX2、ALP和OC的表达,抑制MSC的成骨分化；Poly(I:C)存在的情况下,MSC下调DC成熟表面标志CD40、CD86和MHCⅡ的表达,增强DC对右旋糖酐的吞噬能力,并抑制DC对T的活化作用.结论:Poly(I:C)能够影响MSC的成骨分化及免疫调节功能,提示TLR3信号的活化可能在成骨分化中发挥作用.%Objective:Mesenchymal stem cells (MSCs) can express Toll-like receptors (TLRs) , but it remains unclare on the effect of TLR ligand on MSCs. Here, we studied the impact of the TLR3 ligand-Poly (I:C) on the phenotype, cell viability, apopto-sis, osteoblast differentiation and immune-suppressive functions of MSC. Methods: The mixed enzyme method were used for the isolation and culture of human umbilical cord mesenchymal stem cells ( MSCs). FCM was used to examined the expression of CD29, CD44, CD105, CD31, CD117 and CD45 on MSC with the treatment of TLR3 ligand-Poly (I:C). CCK-8 and Annexin-V/PI were used to measure cell viability and apoptosis with the treatment of a series of concentrations of Poly (I: C ) . Then we investigated the effect of Poly(I:C) on osteoblast differentiation of MSC. FCM was used to measure the expression of CD40, CD86, MHC Ⅱ and the cellular uptake of Dextran of DC, and the allogeneic MLR assay was used to investigate antigen-presenting function of DC both in the presence of Poly(I:C) and MSCs. Results: Poly (I:C) did not affect the phenotype and proliferation of MSC. Only high concentrations of Poly (I:C) caused apoptosis of MSC. Moreover, Poly (I:C) can inhibit the expression of RUNX2, ALP and OC in MSC. In addition, both the expression of CD40, CD86 and MHC Ⅱ were upregulated on DC, the phagocytic capacity of DC was enhanced and the activation on T cells of DC was inhibited in the presence of Poly(I:C) and MSCs. Conclusion; TLR3 ligand-Poly (I:C) could affect the differentiation potential and the immunol-suppressive functions of MSCs.