Zhang J P, Yang H Q, Yan Y, Hou B X
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.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2019 Dec 9;54(12):841-846. doi: 10.3760/cma.j.issn.1002-0098.2019.12.010.
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). 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 . , 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. 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) (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) (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)] (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) (0.01). Western blotting results showed that osteocalcin expression in PRDM9sh was significantly lower than that in control group at 1 and 2 weeks after osteogenesis induction. Animal transplantation experiments results indicated that PRDM9 significantly inhibited the osteogenesis of PDLSC , and the proportion of osteogenic area calculated showed that the osteogenic capacity of PRDM9sh [(3.8±2.41)%] was significantly lower than that in control group [(24.54±7.06)%](<0.05). Depletion of PRDM9 repressed the osteogenic differentiation of stem cells from periodontal ligament and .
为研究组蛋白甲基转移酶之一的PR结构域锌指蛋白9（PRDM9）对牙周膜间充质干细胞（PDLSC）成骨分化能力的影响。使用重组慢病毒载体通过PRDM9 shRNA敲低PRDM9基因的PDLSC被分为PRDM9sh组，转染的shRNA作为对照组。通过逆转录聚合酶链反应（RT-PCR）评估基因表达效率。碱性磷酸酶活性（ALP）、茜素红染色、矿化以及骨钙素，后者属于通过RT-PCR检测的成骨分化标志物，并通过蛋白质印迹法检测牙周膜干细胞的成骨分化能力。将PRDM9sh和对照组细胞移植到背部皮肤以探索成骨情况。通过图像分析软件计算新骨组织的面积百分比并进行统计学分析。RT-PCR结果显示，PRDM9sh组中PRDM9基因的相对表达（0.460±0.017）显著低于对照组（1.000±0.107）（P<0.05）。诱导后5天测定的ALP活性结果显示，PRDM9sh细胞（0.762±0.063）与对照组（1.225±0.058）相比显著降低（P<0.01）。成骨诱导2周和3周的茜素红染色显示，PRDM9sh组的染色明显比对照组浅。定量钙分析结果显示，PRDM9sh组在成骨诱导2周和3周时的钙离子浓度[（0.071±0.004），（0.075±0.001）]显著低于对照组在2周和3周时的浓度[（0.282±0.006），（0.485±0.004）]（P<0.01）。RT-PCR结果显示，PRDM9sh组中骨钙素mRNA的相对表达（1.059±0.148）在2周时显著低于对照组（2.542±0.190）（P<0.01）。蛋白质印迹结果显示，成骨诱导1周和2周后，PRDM9sh组中骨钙素的表达明显低于对照组。动物移植实验结果表明，PRDM9显著抑制PDLSC的成骨，计算得出的成骨面积比例显示，PRDM9sh组的成骨能力[（3.8±2.41）%]显著低于对照组[（24.54±7.06）%]（P<0.05）。PRDM9的缺失抑制了牙周膜干细胞的成骨分化。
