摘要
目的研究骨形态生成蛋白(bone morphogenetic protein,BMP)拮抗剂Gremlin1(GREM1)对根尖牙乳头干细胞(stem cells from the apical papilla,SCAPs)功能的影响,并探索其机制。方法体外分离培养SCAPs,免疫荧光染色实验检测GREM1在SCAPs的亚细胞定位;利用GREM1shRNA慢病毒转染,敲低GREM1;分别对GREM1敲低组和对照组SCAPs进行成骨诱导,实时荧光定量PCR(Real time-PCR)和Western blot检测转染后GREM1的敲低效果。取两组细胞,成骨诱导7 d后进行碱性磷酸酶(ALP)活性测定,成骨/成牙本质诱导3周后进行茜素红染色,并用Real time-PCR检测成骨诱导0周、1周、2周、3周后成骨/成牙相关基因骨钙素(OCN)、骨桥素(OPN)、骨涎蛋白(BSP)、牙本质基质蛋白(DMP1)、牙本质涎磷蛋白(DSPP)以及成骨/成牙关键转录因子Runt相关转录因子2(RUNX2)、成骨细胞特异性转录因子(OSX)、同源盒基因2(DLX2)的表达。取两组细胞,CCK8和流式细胞术检测细胞增殖能力变化;Real time-PCR检测衰老相关基因p53、细胞周期调控因子1(Waf1)和干性相关基因krupple样因子4(KLF4)、性别决定区Y框蛋白2(SOX2)的表达,以及BMPs相关基因(BMP2、BMP4、BMP5、BMP6、BMP7、BMP9)的表达。结果免疫荧光实验表明GREM1在胞核内表达量比胞浆中更高;Real time-PCR和Western blot验证了GREM1敲低组的敲低效果。GREM1敲低组ALP活性高于对照组(P<0.05),茜素红染色浅于对照组;OCN、DMP1在1周、2周、3周时表达均有升高,OPN在2周时升高,BSP在3周时升高,DSPP在1周时升高,差异有统计学意义(P<0.05),成骨关键转录因子RUNX2、OSX、DLX2均在不同时段升高,差异有统计学意义(P<0.05)。CCK-8法和流式细胞术显示GREM1敲低组增殖能力较对照组下降(P<0.05)。与对照组相比,GREM1敲低组BMP2、BMP6、BMP7表达升高,SOX2、KLF4表达升高(P<0.05),P53、Waf1表达下降(P<0.05)。结论GREM1敲低后,能促进SCAPs成骨/成牙分化及干性,抑制SCAPs的增殖及衰老;GREM1对
Objective To study the effect of bone morphogenetic protein(BMP)antagonist Gremlin1(GREM1)on the function of stem cells from apical papilla(SCAPs)and explore its mechanism.Methods After isolation and culturing of stem cells from apical papilla in vitro,immunofluorescent staining was done to examine the subcellular localization of GREM1 in SCAPs.Transfection with lentiviral GREM1 shRNA was done to knock-down the GREM1.The SCAPs were subjected to osteogenic induction in both the GREM1 knockdown group and the control group,and the knockdown effect of GREM1 was examined using real time-PCR and Western blot.Two groups of cells were collected and the alkaline phosphatase(ALP)activity was measured 7 d after osteogenic induction.Alizarin red staining was done 3 weeks after osteogenic/odontogenic induction and real time-PCR was done after 0,1,2,3 weeks of osteogenic induction to examine the expression of osteogenic/odontogenic marker genes,including osteocalcin(OCN),osteopontin(OPN),bone sialoprotein(BSP),dentin matrix protein 1(DMP1),dentin sialophosphoprotein(DSPP)and and the critical transcription factor osterix(OSX),Runt-related transcription factor 2(RUNX2),and distal-less homebox 2(DLX2).Two groups of cells were collected,and CCK-8 and CFSE assay were used to evaluate changes in cell proliferation.In addition,real time-PCR was used to examine the expression of senescence-related genes p53 and wide-type activated factor 1(Waf1),a regulatory factor of the cell cycle,stemness associated gene krupple-like factor 4(KLF4),and SRY related HMG box-2(SOX2),and the expression of bone morphogenetic protein(BMP)2,4,5,6,7,9 after GREM1 knockdown.Results Immunofluorescence staining showed that the expression of GREM1 in the nucleus was higher than that in the cytoplasm.Real time-PCR and Western blot affirmed that GREM1 was knocked down steadily.The ALP activity of the GREM1 knockdown group was higher than that of the control group(P<0.05),and the alizarin red staining was lighter than that of the control group.The expression of O
作者
朱行燕
刁树
杨东梅
范志朋
ZHU Xing-yan;DIAO shu;YANG Dong-mei;FAN Zhi-peng(Department of Dentistry,Beijing Children’s Hospital,Capital Medical University,National Center for Children’s Health,Beijing 100045,China;Department of Pediatric Dentistry,Beijing Stomatological Hospital,Capital Medical University,Beijing 100050,China;Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction,Research Institute of Beijing Stomatological Hospital,Capital Medical University,Beijing 100050,China)
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2021年第3期409-415,共7页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(No.81800923)资助。
关键词
根尖牙乳头干细胞
GREM1
骨形态生成蛋白
成骨/成牙分化
Stem cells from the apical papilla
GREM1
Bone morphogenetic protein
Osteogenic/odontogenic differentiation
