白血病抑制因子通过JAK2/STAT3信号通路对牙周膜细胞增殖影响的研究

The study of leukemia inhibitory factor on proliferation of HPDLCs via JAK2/STAT3 signaling pathway in vitro

目的:探究白血病抑制因子(leukemia inhibitory factor,LIF)及(双面联胎激酶2,Janus kinase 2,JAK2)/(信号传导转录启动因子3,signal transducer and activator of transcription 3,STAT3)对人牙周韧带细胞(human perio-dontal ligament cells,HPDLCs)增殖影响.方法:体外培养HPDLCs至第3代,在LIF、LIF中和抗体及JAK2抑制剂(AG490)刺激后检测HPDLCs增殖情况,HPDLCs分裂速度、细胞周期及对JAK2/STAT3信号通路的影响.结果:CCK-8检测显示,LIF在20~40 ng/mL促进HPDLCs增殖(P<0.01),而LIF中和抗体或AG490组,增殖显著被抑制(P<0.01).流式分析显示LIF组HPDLCs荧光强度最弱而LIF/AG490和AG490组荧光强度较强.碘化丙啶(propidium iodide,PI)染色显示LIF组PI指数明显增高,S期明显增长,而LIF/AG490和AG490组则相反.免疫荧光显示LIF组p-JAK2及-STAT3的表达显著提高.Western Blot显示LIF组JAK2明显磷酸化;而AG490组,p-JAK2被减弱;STAT3的磷酸化也如此.结论:LIF通过JAK2/STAT3信号通路促进HPDLCs增殖.

Objective:To explore the effect of leukemia inhibitory factor(LIF)and LIF/(Janus kinase 2,JAK2)/(signal transducer and activator of transcription 3,STAT3)signaling pathway on proliferation of human periodontal ligament cells(HPDLCs)in vitro.Methods:Experiments were carried out with passage 3 HPDLCs.The HPDLCs were then changed to α-MEM with LIF in different concentrations,and then LIF neutralizing antibodyand AG490 were also added.CCK-8 kit was used to detect the proliferation of HPDLCs.The effects of LIF on cell cycle and cell division of HPDLCs were investigated by fluorescence-activated cell sorting(FACS)using propidium iodide(PI) and 5-(and 6-)carboxy fluorescein diacetate succinimidyl ester(CFSE) staining methods.Immunofluorescence assay and Western Blot were used to examine the expression of p-JAK2 and p-STAT3 of HPDLCs stimulated by LIF.Results:CCK-8 assay showed that LIF promoted the proliferation of HPDLCs at 20 and 40 ng/mL(P<0.01).LIF induced proliferation of HPDLCs were inhibited by LIF neutralizing antibody or AG490.FACS analysis showed that,the weakest fluorescence was detected by CFSE staining after LIF stimulation,and the highest PI index and longest S phase was found by PI staining after LIF stimulation.Immunofluorescence assay showed that faint red fluorescence began to show in the control group,and intensive red fluorescence was found in the cytoplasm of HPDLCs in the group stimulated by LIF.Based on Western Blot analysis,p-JAK2 protein was not detected and p-STAT3 detected slightly in the control group.And protein expression of p-JAK2 and p-STAT3 was dramatically strong after LIF stimulation.But the expression was decreased by adding AG490.Conclusion:LIF promoted proliferation of HPDLC via JAK2/STAT3 signaling pathway in vitro.