中华口腔医学杂志
中華口腔醫學雜誌
중화구강의학잡지
Chinese Journal of Stomatology
2013年
11期
672-678
,共7页
伍丽静%刘路%陈丽虹%韦曦
伍麗靜%劉路%陳麗虹%韋晞
오려정%류로%진려홍%위희
细胞增殖%细胞分化%牙髓细胞%八聚体转录结合因子4A
細胞增殖%細胞分化%牙髓細胞%八聚體轉錄結閤因子4A
세포증식%세포분화%아수세포%팔취체전록결합인자4A
Cell proliferation%Multiple differentiation%Dental pulp cells%Octamer-binding transcription factor 4A
目的 研究八聚体转录结合因子4A(octamer-binding transcription factor 4A,Oct4A)在人牙髓细胞(human dental pulp cells,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(reahi mequantitative PCR,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),50 nmol/L siRNA经脂质体LipofectamineTM RNAiMAX转染DPC 24、48、72、96和120 h,以无义序列-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Oct4A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DPC 14 d后矿化结节形成情况,油红O染色检测成脂诱导DPC 14 d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(dentin sialophosphoprotein,DSPP)和成脂向分化标志物脂蛋白脂酶(lipoprotein lipase,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DPC 72 h,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.
目的 研究八聚體轉錄結閤因子4A(octamer-binding transcription factor 4A,Oct4A)在人牙髓細胞(human dental pulp cells,DPC)的錶達及對細胞增殖、成牙本質嚮和成脂嚮分化能力的影響.方法 實時熒光定量PCR(reahi mequantitative PCR,RT-qPCR)技術檢測健康人DPC中Oct4A的錶達；閤成特異性針對Oct4A的siRNA(Oct4A榦擾組),50 nmol/L siRNA經脂質體LipofectamineTM RNAiMAX轉染DPC 24、48、72、96和120 h,以無義序列-siRNA轉染的DPC作為陰性對照,細胞計數試劑盒法檢測細胞增殖情況、RT-qPCR檢測Oct4A的mRNA水平以選取最佳轉染時間；茜素紅染色檢測成牙本質誘導DPC 14 d後礦化結節形成情況,油紅O染色檢測成脂誘導DPC 14 d後脂滴形成情況,RT-qPCR技術檢測成牙本質嚮分化標誌物牙本質涎燐蛋白(dentin sialophosphoprotein,DSPP)和成脂嚮分化標誌物脂蛋白脂酶(lipoprotein lipase,LPL)的mRNA錶達變化；蛋白質印跡法檢測DSPP和LPL的錶達.結果 Oct4A在第3代DPC中錶達最高(2.10±0.10),為第1代DPC的2.10倍(與第1、7代相比P =0.000),之後隨細胞傳代錶達下降；Oct4A-siRNA轉染DPC 72 h,榦擾效率達峰值(69.7％)；與正常細胞組及陰性對照組相比Oct4A榦擾組細胞增殖能力顯著下降,礦化結節和脂滴形成顯著減少(P =0.000),分化標誌物DSPP和LPL錶達量顯著下降(P =0.000).結論 瞬時榦擾牙髓細胞中Oct4A的錶達可以降低細胞增殖和成牙本質嚮、成脂嚮分化能力.
목적 연구팔취체전록결합인자4A(octamer-binding transcription factor 4A,Oct4A)재인아수세포(human dental pulp cells,DPC)적표체급대세포증식、성아본질향화성지향분화능력적영향.방법 실시형광정량PCR(reahi mequantitative PCR,RT-qPCR)기술검측건강인DPC중Oct4A적표체；합성특이성침대Oct4A적siRNA(Oct4A간우조),50 nmol/L siRNA경지질체LipofectamineTM RNAiMAX전염DPC 24、48、72、96화120 h,이무의서렬-siRNA전염적DPC작위음성대조,세포계수시제합법검측세포증식정황、RT-qPCR검측Oct4A적mRNA수평이선취최가전염시간；천소홍염색검측성아본질유도DPC 14 d후광화결절형성정황,유홍O염색검측성지유도DPC 14 d후지적형성정황,RT-qPCR기술검측성아본질향분화표지물아본질연린단백(dentin sialophosphoprotein,DSPP)화성지향분화표지물지단백지매(lipoprotein lipase,LPL)적mRNA표체변화；단백질인적법검측DSPP화LPL적표체.결과 Oct4A재제3대DPC중표체최고(2.10±0.10),위제1대DPC적2.10배(여제1、7대상비P =0.000),지후수세포전대표체하강；Oct4A-siRNA전염DPC 72 h,간우효솔체봉치(69.7％)；여정상세포조급음성대조조상비Oct4A간우조세포증식능력현저하강,광화결절화지적형성현저감소(P =0.000),분화표지물DSPP화LPL표체량현저하강(P =0.000).결론 순시간우아수세포중Oct4A적표체가이강저세포증식화성아본질향、성지향분화능력.
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 Oct4A in DPC was detectedby realtime quantitative PCR(RT-qPCR).siRNA-Oct4A was constructed and transfected(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 RTqPCR 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 Oct4A significantly downregulated the cell proliferation rate and multilineage differentiation capability of DPC.
