牙周膜干细胞调节巨噬细胞功能的体外研究

Periodontal ligament stem cells regulate the functions of macrophages in vitro

目的探讨牙周膜干细胞(PDLSCs)对外周血来源巨噬细胞的表型和功能的体外影响。方法分离、培养PDLSCs,并检测其间充质干细胞标志物基质细胞抗原-1(STRO-1)、表面抗原146(CD146)、表面抗原90(CD90)的表达情况及骨向、脂肪向分化能力。分离外周血来源的巨噬细胞。将PDLSCs与等量异体巨噬细胞在Transwell培养系统中37℃、5%CO2条件下共培养,为实验组。巨噬细胞的单独培养设置为对照组。共培养3 d后,提取巨噬细胞,采用流式细胞术检测CD14+CD206+巨噬细胞的表达情况;共培养24 h后,提取巨噬细胞,加入荧光素异硫氰酸酯标记的葡聚糖,孵育30 min后,采用流式细胞术检测巨噬细胞的吞噬率;共同培养3 d后,取细胞培养上清,采用酶联免疫吸附法检测上清中白介素-10(IL-10)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的浓度。结果PDLSCs呈梭状的成纤维细胞样,表达STRO-1、CD146和CD90,可以骨向和脂肪向分化。与对照组相比:(1)共培养组的CD14+CD206+巨噬细胞表达率明显升高[(38.73±6.32)%vs(8.39±2.71)%,t=127.7,P=0.0049];(2)共培养组的巨噬细胞吞噬率无显著变化[(36.7±5.1)%vs(38.6±4.3)%,t=3.904,P=0.1596];(3)共培养组的IL-10浓度明显升高[(382.5±18.2)pg/mL vs(198.5±11.4)pg/mL,t=76.36,P=0.0003],IL-6浓度显著降低[(453.1±70.42)pg/mL vs(936.7±49.9)pg/mL,t=53.12,P=0.0115],TNF-α浓度也显著降低[(64.9±11.3)pg/mL vs(131.7±19.3)pg/mL,t=51.48,P=0.0006]。结论PDLSCs可以促使巨噬细胞向M2型极化,不影响巨噬细胞的吞噬功能,促进抗炎因子IL-10的分泌,抑制炎性因子IL-6和TNF-α的分泌。

Objective To explore the effects of periodontal ligament stem cells(PDLSCs)on the phenotypes and functions of macrophages.Methods After PDLSCs were isolated and cultured,the expression profiles of STRO-1,CD146 and CD90,as well as the multipotent differentiation capabilities were detected.After macrophages were isolated from peripheral blood,they were cocultured with an equal amount PDLSCs in Transwell co-culture condition at 37℃and 5%CO2,which were set as the experimental group.Macrophages cultured alone were set as the control group.After 3 d co-culture,the expression profiles of CD14+CD206+macrophages were examined by flow cytometry.After 24 h co-culture,macrophages were obtained,fluorescein isothiocyanate labeled dextran was added.Then,after 30 min incubation,the phagocytosis rate of macrophages was detected with flow cytometry.After 3 d co-culture,the supernatant was collected,and the concentrations of IL-10,IL-6 and TNF-αwere determined with enzyme-linked immunosorbent assays.Results PDLSCs displayed fusiform fibroblast-like morphology,positive for the mesenchymal stem cells surface markers including STRO-1,CD146 and CD90,and could differentiate into bone cells and lipid cells.Compared with the control group,the experimental group had significantly increased expression of CD14+CD206+macrophages[(38.73±6.32)%vs(8.39±2.71)%,t=127.7,P=0.0049),unchanged phagocytosis rate of macrophages[(36.7±5.1)%vs(38.6±4.3)%,t=3.904,P=0.1596],elevated level of IL-10[(382.5±18.2)pg/mL vs(198.5±11.4)pg/mL,t=76.36,P=0.0003],but decreased levels of IL-6[(453.1±70.42)pg/mL vs(936.7±49.9)pg/mL,t=53.12,P=0.0115)and TNF-α[(64.9±11.3)pg/mL vs(131.7±19.3)pg/mL,t=51.48,P=0.0006].Conclusion PDLSCs are capable of converting macrophages into M2 phenotype without affecting the phagocytic functions.Meanwhile,they can stimulate the secretion of IL-10 but inhibit the secretion of IL-6 and TNF-α.