肿瘤坏死因子α刺激下人牙髓干细胞与CD3+T细胞共培养后生物学功能的变化

Changes of biological function of human dental pulp stem cells co-cultured with CD3+T cells stimulated by TNF-α

背景牙髓干细胞(dental pulp stem cells,DPSCs)作为目前组织工程中优质的种子细胞,具有强大的免疫调节能力。在炎症微环境下,牙髓干细胞的多种生物学功能会有不同程度的改变。目的研究肿瘤坏死因子α(tumour necrosis factor-α,TNF-α)刺激下人牙髓干细胞与CD3+T细胞共培养后生物学功能变化。方法劈冠法分离获取DPSCs并进行分离传代。利用5 ng/mL、10 ng/mL、15 ng/mL不同浓度的外源性TNF-α诱导P3代细胞产生炎症反应,CCK8法检测各组细胞增殖情况;对正常组织来源的牙髓干细胞(DPSCs obtained from a healthy microenvironment,HDPSCs)及炎症微环境来源(10 ng/mL的TNF-α预刺激24 h)的牙髓干细胞(DPSCs obtained from a inflammatory microenvironment,IDPSCs)进行成骨诱导分化培养并进行茜素红染色,同时对HDPSCs进行成脂诱导分化培养并进行油红O染色。实时荧光定量PCR(quantitative real-time PCR,qPCR)检测HDPSCs和IDPSCs的TNF-α、白细胞介素-1β(interleukin-1β,IL-1β)的mRNA表达水平;实时定量qPCR检测常规培养(undifferentiation,undiff)和成骨诱导(differentiation,diff)前后成骨基因Runt相关因子2(runt-related gene 2,Runx2)、碱性磷酸酶(alkaline phosphatase,ALP)的mRNA表达水平。免疫磁珠法从人外周血中分离获取CD3+T细胞,将DPSCs和CD3+T细胞按照1∶0、1∶10、1∶20、1∶50、1∶100的比例在正常环境及TNF-α刺激环境中直接共培养,用qPCR分别检测TNF-α、IL-1β、β-catenin及淋巴样增强因子1(lymphoid enhancer factor 1,LEF-1)的mRNA表达水平。结果CCK8法示10 ng/mL TNF-α刺激组的DPSCs吸光度值显著高于其他三组(P<0.05)。常规培养条件下,TNF-α刺激组Runx2、ALP的mRNA表达水平与正常组无统计学差异;成骨诱导条件下成骨细胞相关的mRNA表达正常组低于TNF-α刺激组(P<0.05)。IDPSCs组IL-1β、TNF-α的mRNA表达高于HDPSCs组(P<0.05)。在DPSCs与CD3+T细胞共培养实验中,正常组DPSCs与CD3+T培养比例为1∶100时IL-1β、LEF-1的mRNA表达水平最低,β-catenin的mRNA表达水平最高,1∶20时TNF-α的mRNA表达水平最低;而在TNF-α刺激组中,比例为1∶20时IL-1β、TNF-α的mRNA表达水平最低,β-catenin及LEF-1的mRNA表达水平最高。结论在TNF-α刺激下,DPSCs的炎性因子表达增加,促进向成骨细胞分化;在与CD3+T细胞共培养的免疫环境中,炎性因子受到抑制,Wnt/β-catenin经典通路被激活。

Background Dental pulp stem cells,as high-quality seed cells in tissue engineering,have strong immunomodulatory ability.In the inflammatory microenvironment,the biological functions of dental pulp stem cells will change in different degrees.Objective To study the functional changes of human pulp stem cells(DPSCs)co-cultured with CD3+T cells under the stimulation of tumor necrosis factor-α(TNF-α).Methods DPSCs were isolated by crown splitting method and subcultured.Different concentrations of exogenous TNF-αwere added for inducing inflammation in P3 cells.The proliferations of cells were detected by CCK8 assay.The messenger RNA(mRNA)expression levels of TNF-αand interleukin-1β(IL-1β)in control DPSCs obtained from healthy microenvironment(HDPSCs)group and DPSCs obtained from inflammatory microenvironment(IDPSCs)group were detected by real-time PCR(RT-PCR).The mRNA levels of osteogenic genes Runt-related gene 2(Runx2)and alkaline phosphatase(ALP)before and after conventional culture(undiff)and osteogenic induction(diff)were detected by RT-PCR.CD3+T cells were isolated from human peripheral blood by immunomagnetic beads method.CD3+T cells and DPSCs were co-cultured with or without TNF-αstimulation at the ratio of 1:10,1:20,1:50 and 1:100,respectively.The mRNA expression levels of TNF-α,IL-1β,β-catenin and LEF-1 in different groups were detected by RT-PCR.Results At 7 d,compared with the control group,the blank group,the 5 ng/mL TNF-αgroup and 15 ng/mL TNF-αgroup,the DPSCs treated by 10 ng/mL TNF-αshowed higher proliferation rate(P<0.05).After osteogenic induction,the mRNA expression levels of Runx2 and ALP were significantly higher in the TNF-αstimulated group than those in the control group.Under the osteogenic differentiation culturing condition,the expressions of osteogenic genes in the control group were lower than those in the TNF-αstimulated group(P<0.05).The inflammatory factors mRNA levels of IL-1βand TNF-αin DPSCs stimulated by TNF-αwere significantly higher than those in control group(P<0.05).DPSCs were co-cultured with CD3+T cells at different ratio from 1∶10 to 1∶100.In the control group,mRNA expressions of IL-1β,LEF-1 andβ-catenin showed the lowest level at the co-culture ratio of 1∶100,and the mRNA expression of TNF-αwas the lowest at 1∶20.In the TNF-αstimulated group,the mRNA expression of IL-1βand TNF-αwere the lowest when the co-culture ratio was 1∶20,but the mRNA expression levels ofβ-catenin and LEF-1 were the highest compared with other co-culturing conditions.Conclusion Under TNF-αstimulation,the expression of inflammatory cytokines and osteogenic ability in DPSCs increases.In the immune environment,when co-cultured with CD3+T cells,the development of inflammation is inhibited and the Wnt/β-catenin classical pathway is activated.