干扰八聚体转录结合因子4A对人牙髓细胞增殖与多向分化能力的影响  

Interference of octamer-binding transcription factor 4A and its effect on the proliferation andmultiple differentiation of human dental pulp cells

作　　者：伍丽静 刘路[1] 陈丽虹[1] 韦曦[1] 

机构地区：[1]中山大学光华口腔医学院·附属口腔医院牙体牙髓科·广东省口腔医学重点实验室,广州510055

出　　处：《中华口腔医学杂志》2013年第11期672-678,共7页Chinese Journal of Stomatology

基　　金：国家自然科学基金（81070830）;广东省科技计划（20118050300007）;广东省自然科学基金（$2012040008476）;广东省研究生创新培养计划（sybzzxm201019）

摘　　要：目的研究八聚体转录结合因子4A（octamer—bindingtranscriptionfactor4A，Oct4A）在人牙髓细胞（humandentalpulpcells，DPC）的表达及对细胞增殖、成牙本质向和成脂向分化能力的影响。方法实时荧光定量PCR（reahimequantitativePCR，RT—qPCR）技术检测健康人DPC中Oct4A的表达；合成特异性针对Oct4A的siRNA（Oct4A干扰组），50nmol／LsiRNA经脂质体Lipofectamine。TMRNAiMAX转染DPC24、48、72、96和120h，以无义序列一siRNA转染的DPC作为阴性对照，细胞计数试剂盒法检测细胞增殖情况、RT—qPCR检测OctdA的mRNA水平以选取最佳转染时间；茜素红染色检测成牙本质诱导DPC14d后矿化结节形成情况，油红0染色检测成脂诱导DPC14d后脂滴形成情况，RT—qPCR技术检测成牙本质向分化标志物牙本质涎磷蛋白（dentinsial叩hosph叩rofein，DSPP）和成脂向分化标志物脂蛋白脂酶（1ipoproteinlipase，LPL）的mRNA表达变化；蛋白质印迹法检测DSPP和LPL的表达。结果Oct4A在第3代DPC中表达最高（2．10±0．10），为第1代DPC的2．10倍（与第1、7代相比P=0．000），之后随细胞传代表达下降；Oct4A—siRNA转染DPC72h，干扰效率达峰值（69．7％）；与正常细胞组及阴性对照组相比Oct4A干扰组细胞增殖能力显著下降，矿化结节和脂滴形成显著减少（P=0．000），分化标志物DSPP和LPL表达量显著下降（P=0．000）。结论瞬时干扰牙髓细胞中Oct4A的表达可以降低细胞增殖和成牙本质向、成脂向分化能力。Objective To investigate the expression of octamer-binding transcription factor 4A （Oct4A） in human dental pulp cells （DPC）and the effect of Oct4A on the proliferation and multiple differentiation ability of DPC. Methods Expression of Oet4A in DPC was detectedby reahime quantitative PCR（RT-qPCR）. siRNA-Oct4A was constructed and transfeeted（50 nmol/L） into DPC with LipofectamineTM RNAiMAX for 24,48,72,96 and 120 h. The proliferation rate of DPC was examined using cell counting kit 8 （ CCK-8 ） assay. The alizarin red staining was used to observe the formation of calcification nodules in DPC with 14 d of osteogenic induction, and oil red O staining to observe the formation of lipid droplet in DPC with 14 d of adipogenic induction. The expression of osteogenesis-related genes dentin sialophosphoprotein（DSPP） and adipogenesis-related genes lipoprotein lipase（LPL） was detected using RT- qPCR and Western blotting. Results The expression of Oct4A reached the peak in P3 DPC （2. 10 _＋ 0. 10） , which was 2. 10 times as much as that in P1 （P = 0. 000 vs. P1 and P7） ,and decreased along passages. The interference efficiency of DPC transfection peaked at 72 h （ 69. 7% ）. Compared with control group and negative control group （ IR-siRNA ） , the proliferation rate and multiple differentiation ability of DPC in interference group （Oct4A-siRNA）were downregulated （P = 0. 000 ）, and DSPP and LPL in DPC from interference group significantly decreased （P = 0. 000 ）. Conclusions The interference of Oet4Asignificantly downregulated the cell proliferation rate and multilineage differentiation capability of DPC.

关 键 词：细胞增殖 细胞分化 牙髓细胞 八聚体转录结合因子4A 

分 类 号：R780.2[医药卫生—口腔医学]