敲除组蛋白甲基转移酶PR结构域锌指蛋白9抑制牙周膜干细胞成骨向分化  

Depletion of PRDM9 inhibited the osteogenic differentiation potential of periodontal ligament stem cells

作　　者：张建鹏[1] 杨昊清 严妍 侯本祥[1] Zhang Jianpeng;Yang Haoqing;Yan Yan;Hou Benxiang(Department of Endodontics,School of Stomatology,Capital Medical University,Beijing 100050,China;Beijing Key Laboratory of Tooth Regeneration and Function Reconstruction,Institute of Dental Research,School of Stomatology,Capital Medical University,Beijing 100050,China)

机构地区：[1]首都医科大学口腔医学院牙体牙髓科,100050 [2]首都医科大学口腔医学院口腔医学研究所全牙再生与口腔组织功能重建北京市重点实验室,100050

出　　处：《中华口腔医学杂志》2019年第12期841-846,共6页Chinese Journal of Stomatology

基　　金：北京市教育委员会科技发展计划一般项目(KM201810025026)。

摘　　要：目的研究组蛋白甲基转移酶PR结构域锌指蛋白9(PR domain zinc finger protein 9,PRDM9)对牙周膜干细胞(periodontal ligament stem cell,PDLSC)成骨向分化的影响.方法将与PRDM9序列互补的短发夹RNA亚克隆到含有绿荧光蛋白的慢病毒载体中,利用重组慢病毒敲除PDLSC中的PRDM9基因,形成敲除组(简称PRDMsh组),转染空载体sh RNA作为对照组;应用反转录PCR(reverse transcription PCR,RT-PCR)检测PRDM9基因的敲除效率;通过碱性磷酸酶(alkalinephosphatase,ALP)活性测定、茜素红染色和钙离子定量分析检测敲除组PRDM9sh与对照组细胞成骨能力的差异;通过实时定量RT-PCR及蛋白免疫印迹实验检测敲除组PRDM9sh和对照组成骨分化指标骨钙蛋白在mRNA水平及蛋白水平的表达差异;通过裸鼠皮下移植物HE染色比较敲除组PRDM9sh及对照组体内成骨能力,计算新成骨组织面积百分比并进行统计学分析.结果实时定量RT-PCR结果显示,敲除组PRDM9sh中PRDM9基因的相对表达量(0.460±0.017)显著低于对照组(1.000±0.107)(P<0.05);成骨诱导5d后,敲除组PRDM9sh的ALP活性(0.762±0.063)显著低于对照组(1.225±0.058)(P<0.01);成骨诱导2、3周后,茜素红染色结果显示,与对照组相比敲除组PRDM9sh染色明显变浅;成骨诱导2、3周后,敲除组PRDM9sh钙离子含量[分别为(0.071±0.004)、(0.075±0.001)]较对照组[分别为(0.282±0.006)、(0.485±0.004)]显著降低(P<0.01);RT-PCR结果显示成骨2周时,敲除组PRDM9sh骨钙蛋白mRNA水平的相对表达量(1.059±0.148)显著低于对照组(2.542±0.190)(P<0.01);蛋白质印迹法结果显示成骨诱导1、2周时,敲除组PRDM9sh骨钙蛋白的蛋白水平表达明显弱于对照组;裸鼠皮下移植物HE染色及计算新成骨组织面积百分比结果显示敲除组PRDM9sh新成骨组织面积百分比[(3.8±2.41)%]较对照组[(24.54±7.06)%]显著降低(P<0.05).结论敲除PRDM9基因可以抑制牙周膜干细胞的体内外成骨分化功能.Objective To investigate the effect of PR domain zinc finger protein 9(PRDM9),one of the histone methylated transferases,on osteogenic differentiation ability of periodontal ligament mesenchymal stem cells(PDLSC).Methods PDLSC with PRDM9 gene knocked down by PRDM9 shRNA using recombinant lentiviral vector were allocated into the PRDM9sh group,and the transfected shRNA was as the control group.The gene expression efficiency was evaluated by reverse transcription polymerase chain reaction(RT-PCR).Alkaline phosphatase activity(ALP),alizarin red staining,mineralization and osteocalcin,which belongs to osteogenic differentiation markers detected by RT-PCR and Western blotting to detect the osteogenic differentiation ability of stem cells from periodontal ligaments in vitro.In vivo,PRDM9sh and control group cells was transplanted into the dorsal dermal to explore the osteogenesis.The area percentage of new osteogenic tissue was calculated by image pro software and statistically analyzed.Results RT-PCR results showed that the relative expression of PRDM9 gene in PRDM9sh(0.460±0.017)was significantly lower than that in control group(1.000±0.107)(P<0.05).The results of ALP activity determined at 5 days postinduction in a significant decrease in PRDM9sh cells(0.762±0.063)compared with control group(1.225±0.058)(P<0.01).Alizarin red staining induced by osteogenesis at 2 weeks and 3 weeks showed that the staining of PRDM9sh was significantly lighter than that in control group.Quantitative calcium analysis results showed that the calcium ion concentration induced by osteogenesis at 2 weeks and 3 weeks[(0.071±0.004),(0.075±0.001)]in PRDM9sh was significantly lower than that in control group at 2 weeks and 3 weeks[(0.282±0.006),(0.485+0.004)](P<0.01).RT-PCR results showed that the relative expression of osteocalcin mRNA in PRDM9sh(1.059±0.148)was significantly lower than that in control group at 2 weeks(2.542±0.190)(P<0.01).Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower t

关 键 词：甲基转移酶类 牙周膜 间充质干细胞 PR结构域锌指蛋白9 成骨分化 

分 类 号：R73[医药卫生—肿瘤]