炎症状态下A20基因沉默对人牙髓干细胞增殖及分化的影响  

The effects of A20-knockdown on the proliferation and differentiation of human dental pulp stem cells in the inflammatory environment

作　　者：张玉琳 邢金良[2] 仇珺 辛越[1,3] 孙汉堂(指导) 

机构地区：[1]军事口腔医学国家重点实验室,陕西省口腔医学重点实验室,第四军医大学口腔医院牙体牙髓病科,陕西西安710032 [2]第四军医大学基础医学部教学实验中心,陕西西安710032 [3]解放军第二〇三医院,黑龙江齐齐哈尔161000

出　　处：《牙体牙髓牙周病学杂志》2016年第5期261-266,共6页Chinese Journal of Conservative Dentistry

基　　金：国家自然科学基金(81070832);第四军医大学青年英才支持计划Ⅰ类项目(2011)

摘　　要：目的:探讨在体外模拟炎症状态下,A20基因沉默对人牙髓干细胞(h DPSCs)增殖及分化能力的影响。方法:用脂多糖(LPS)刺激h DPSCs,Western-Blot技术检测不同时间点锌指蛋白A20表达量的变化;通过RNA干涉技术构建A20基因沉默的细胞模型,CCK-8法检测细胞增殖能力;矿化液诱导培养后,用碱性磷酸酶检测试剂盒测定ALP的活性,茜素红染色观察矿化结节的形成,并用RT-PCT检测成牙/成骨分化相关基因ALP、DSPP、OCN、RUNX2表达水平。结果:h DPSCs在LPS刺激2 h后,A20的表达即显著增加,在接近72 h时其表达量恢复至基础水平;在模拟炎症状态下,与对照组相比,A20基因沉默后的h DPSCs,其增殖能力明显降低(P<0.05),ALP的活性、矿化结节的形成、成牙/成骨分化相关基因的表达水平均受到抑制(P<0.05)。结论:在炎症状态下,A20在早期呈现一过性的高表达,A20基因沉默能够抑制h DPSCs的增殖及分化能力。AIM： To investigate the effects of A20- knockdown on the proliferation and differentiation of human dental pulp stem cells（ h DPSCs） in the inflammatory environment in vitro. METHODS： h DPSCs were treated by lipopolysaccharide（ LPS）,and the expression of zinc finger protein A20 was detected by Western Blot at different time points. RNAi was used to establish A20 gene silence cell model and CCK-8 assay was applied to assess cell proliferation. After mineralization induction culture,alkaline phosphatase（ ALP） assay kit was used to detect the activity of ALP,alizarin red staining was used to observe the mineral nodules,the m RNA expression of ALP,osteonectin（ OCN）,dentin sialophospho protein（ DSPP） and runt- related transcription factor 2（ RUNX2） was determined by RT- PCR.RESULTS： After 2 h treatment with LPS,A20 expression in h DPSCs increased dramatically,and returned to the basal level after 72 h. In the inflammatory environment,after A20 gene was knocked down,the proliferation of h DPSCs was inhibited（ P〈0. 05）. ALP activity and the formation of mineral nodule were reduced,the m RNA expression of ALP,DSPP,OCN and RUNX2 was down- regulated（ P〈0. 05）. CONCLUTION： In the inflammatory environment,the expression of A20 increased rapidly and temporarily. The knock- down of A20 can inhibit the proliferation,osteogenic and dentinogenic differentiation of h DPSCs.

关 键 词：锌指蛋白A20 人牙髓干细胞(hDPSCs) 脂多糖(LPS) 增殖 分化 

分 类 号：R780.2[医药卫生—口腔医学]