PolyI:C对人脐血内皮祖细胞数量及功能的影响

Effect of PolyI:C on Quantity and Activity of Endothelial Progenitor Cells From Human Umbilical Cord Blood

目的观察Toll样受体3的配体聚肌胞(PolyI:C)对人脐血内皮祖细胞增殖、凋亡及炎性细胞因子表达的影响。方法采用密度梯度离心法获取人脐静脉血单个核细胞,EBM-2细胞培养基进行培养,诱导单个核细胞向内皮祖细胞分化。以不同浓度的PolyI:C(0、0.01、0.1、1 g/L和10 g/L)干预人脐血内皮祖细胞,通过CCK-8细胞增殖试验检测PolyI:C对内皮祖细胞增殖的影响,流式细胞术检测PolyI:C对细胞凋亡的影响。通过逆转录聚合酶链反应对内皮祖细胞表达的Toll样受体进行检测,并检测不同浓度的PolyI:C对内皮祖细胞表达Toll样受体3、炎性细胞因子的影响。结果静息状态下,内皮祖细胞表达较高水平的Toll样受体1、Toll样受体3、Toll样受体4、Toll样受体6,表达较低水平的Toll样受体2、Toll样受体5、Toll样受体7、Toll样受体8、Toll样受体10,不表达Toll样受体9。而PolyI:C能上调Toll样受体3 mRNA表达,并呈量效关系。与对照组相比,较高浓度PolyI:C(1 g/L和10 g/L)持续作用于脐血内皮祖细胞3天后显著抑制内皮祖细胞增殖(P<0.01),终浓度10 g/L的PolyI:C呈时间依赖性抑制内皮祖细胞增殖,且PolyI:C呈剂量依赖性诱导内皮祖细胞凋亡。同时,PolyI:C呈剂量依赖性上调炎性细胞因子白细胞介素1β、白细胞介素6、白细胞介素8、肿瘤坏死因子α、干扰素β的基因表达。结论 PolyI:C可能通过活化Toll样受体3诱导内皮祖细胞凋亡,从而抑制内皮祖细胞增殖,并促进内皮祖细胞表达相关炎性细胞因子。

Aim To investigate the effect of a synthetic dsRNA analog polyriboinosinic polyribocytidylic acid（PolyI：C） on quantity and activity of endothelial progenitor cells from human umbilical cord blood in vitro. Methods Mononuclear cells were isolated from human cord blood by Ficoll density gradient centrifugation and then the cells were cultured in EBM-2 medium to differentiate into endothelial progenitor cells.Endothelial progenitor cells were identified by demonstrating the expression of characteristic cell surface marker CD34,CD133 and KDR by reverse transcriptase polymerase chain reaction.CCK-8 reagent kit was used to analyze the effect of PolyI：C on the proliferation of endothelial progenitor cells.Flow cytometry was used to detect the apoptosis caused by PolyI：C.Reverse transcriptase polymerase chain reaction was performed to detect firstly the expression of Toll-like receptors in endothelial progenitor cells,secondly the expression of Toll-like receptor 3 and inflammatory cytokines induced by PolyI：C. Results Under basal condition,except Toll-like receptor 9,Toll-like receptor 1-10 were expressed in endothelial progenitor cells with high expression levels of Toll-like receptor 1,Toll-like receptor 3,Toll-like receptor 4,Toll-like receptor 6 and low expression levels of Toll-like receptor 2,Toll-like receptor 5,Toll-like receptor 7,Toll-like receptor 8,Toll-like receptor 10.Expression of Toll-like receptor 3 mRNA was upregulated by PolyI：C and there was dose-effect relationship in experiment range.Compared with thecontrol group,PolyI：C groups（1 g/L and 10 g/L） significantly inhibited the proliferation of endothelial progenitor cells and the inhibition of PolyI：C（10 g/L） on endothelial progenitor cells proliferation was time-dependent.PolyI：C induced the apoptosis of endothelial progenitor cells in dose-dependent manner.PolyI：C also upregulated the mRNA expression of inflammatory cytokines such as interleukin-1β,interleukin-6,interleukin-8,tumor necrosis factor-α,interferon-β. Conclusion Poly I：C inhibits the proliferation of endothelial progenitor cells via induction of cell apoptosis,simultaneously up regulates the expression of inflammatory cytokines probably by activating Toll-like receptor 3.