低浓度牙龈卟啉单胞菌脂多糖刺激骨唾液酸蛋白的基因表达和转录

目的研究低浓度牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）脂多糖（Lipopolysaccharide,LPS）对成骨细胞系（ROS17/2.8）中骨唾液酸蛋白（Bonesialoprotein,BSP）基因表达和转录的调节作用,以及细胞外信号调节蛋白激酶（ERK1/2）转导途径阻断剂（U0126）对此调节作用的影响。方法0.01mg/LP.g·LPS作用ROS17/2.8细胞0h、3h、6h和12h后,用Northern杂交观察BSP和骨桥蛋白（Osteopontin,OPN）mRNA的表达;再将ROS17/2.8细胞随机分为四组：空白对照组、LPS（0.01mg/LP.g·LPS）组、U0126（5μmol/L）组、U0126＋LPS组（U0126预刺激细胞30min后,U0126与P.g·LPS共同作用）,各组持续作用12h后,用瞬时转染法分析BSP基因启动子的转录活性。结果0.01mg/LP.g·LPS作用ROS17/2.8细胞12小时后BSP和OPN的mRNA杂交条带增强;0.01mg/LP.g·LPS使BSP基因启动子（pLUC3）转录活性值与空白载体活性值的比值升高1.582（F=5.734,P〈0.05）,U0126使其降低2.693（F=11.500,P〈0.01）,U0126使LPS对比值的升高变化降低2.242（F=6.204,P〈0.05）。结论低浓度（0.01mg/L）P.g·LPS增强ROS17/2.8细胞BSP基因表达和转录,而且其对BSP基因转录活性的上调作用是经由ERK1/2信号转导路径介导的。     

小鼠牙本质涎磷蛋白5′端非编码区启动子报告基因载体的构建和分析

目的：克隆小鼠牙本质涎磷蛋白(dentin sialophosphopmtein，DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性：方法：PCR、报告基因载体构建、瞬时转染和报告基因检测。结果：PCR获得DSPP启动子的3个不同片段，将它们克隆到萤火虫荧光素酶报告基因载体pG13-Enhancer，构建出3种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

小鼠牙本质涎磷蛋白启动子报告基因载体的构建和启动子活性分析

目的：克隆小鼠牙本质涎磷蛋自（dentin sialophosphopmtein,DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性。方法：细胞基因组提取，PCR，瞬时转染和报告基因检测。结果：从MDPC-23细胞基因组中克隆出长为1.5kbp的DSPP启动子，将启动子酶切成不同的片断，克隆到虫工业基础光素酶报告基因载体pC1.3-Enhancer,构建出4种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

Biological effects of cementum and bone extracts on human periodontal fibroblasts

BACKGROUND: Non-collagenous proteins of mineralized tissues play important roles in bone induction during mineralization and in regulating the activity of many types of mesenchymal cells. This study was conducted to determine the effects of acetic acid extracts of bone and cementum on alkaline phosphatase (ALPase) activity and in vitro mineralization of cultured human periodontal fibroblasts (hPF). METHODS: Alveolar bone and cementum obtained from clinically healthy subjects were extracted by a solution containing 0.5 M acetic acid and enzyme inhibitors. Osteoblastic phenotypes of hPF were assayed by ALPase activity, gene expression of bone marker proteins, and the ability to produce in vitro mineralization in culture media containing 50 microg/ml ascorbic acid, 10 mM sodium beta-glycerophosphate, and 10(-7) M dexamethasone. The effects of cementum and bone extracts on the expression of osteoblastic phenotypes in hPF were also determined. RESULTS: Many protein components, varying in molecular weight from 10 to 14 to 120 kDa, were detectable in 10% SDS-PAGE of both cementum and alveolar bone extracts. The hPF cells were found to exhibit a moderate ALPase activity when compared with rat osteosarcoma (ROS) 17/2.8 cells under the same experimental conditions. Gene expression for ALPase, osteocalcin bone sialoprotein, osteopontin, and BMP-7 at mRNA message was detected by RT-PCR in hPF and ROS 17/2.8 cells. The confluent hPF and ROS 17/2.8 cells showed evidence of calcium deposition in the extracellular milieu at 30 and 15 to 30 days' cultures, respectively, under a mineralization medium. The hPF appeared to form mineralized foci with morphological characteristics different from the mineralized nodules produced by ROS 17/2.8 cells. The addition of low concentrations (5 microg/ml) of either cementum or bone extract produced an increase in the size and number of mineralization spots, as well as greater ALPase activity in both hPF and ROS 17/2.8 cultures during the observation periods. CONCLUSIONS: These results suggest that hPF possess certain mineralizing phenotypes, and that acetic acid extracts of bone and cementum contain components capable of stimulating osteogenic differentiation of hPF.     

微小RNA-17在糖基化终末产物刺激下人牙周膜干细胞骨向分化过程中的调控作用

目的 检测微小RNA-17（mir-17）在糖基化终末产物（AGEs）刺激下人牙周膜干细胞（HPDLSCs）骨向分化过程中的表达,并分析其对该过程的影响。方法 体外组织块法和有限稀释法克隆化培养HPDLSCs。实时定量聚合酶链反应（real time PCR）检测实验组细胞在骨向分化过程中不同时间点mir-17的表达;采用细胞转染技术过表达和抑制mir-17的表达,real time PCR和Western blot分别检测转染前后其成骨基因mRNA水平和蛋白水平的表达情况。结果 成骨诱导3、7、14 d后,对照组和实验组mir-17的表达均下调,差异有统计学意义（P<0.05）。上调mir-17后,与对照组相比,实验组骨涎蛋白（BSP）、碱性磷酸酶（ALP）、Runt相关转录因子-2（Runx-2）mRNA表达水平以及Runx-2蛋白水平均明显降低;下调mir-17后,实验组BSP、ALP、Runx-2 mRNA表达水平以及Runx-2蛋白水平均高于对照组。结论 AGEs通过影响HPDLSCs骨向分化过程中mir-17的表达从而抑制了HPDLSCs的骨向分化。     

转录因子c-Jun和c-Fos在牙本质涎磷蛋白基因表达中的作用

目的 研究成牙本质细胞内转录因子c-Jun和c-Fos在牙本质涎磷蛋白（DSPP）基因转录调控中的作用, 探索成牙本质细胞内DSPP基因表达的调控机制。方法 细胞免疫组化观察MDPC-23细胞内c-Jun和c-Fos蛋白分子的表达。瞬时转染和报告基因检测c-Jun和c-Fos在DSPP基因转录中的作用。结果 MDPC-23细胞表达c-Jun和c-Fos蛋白,c-Jun和c-Fos主要分布在MDPC-23细胞胞核。c-Jun或c-Fos过表达均显著抑制DSPP基因启动子活性。结论 证实成牙本质细胞内转录因子c-Jun和c-Fos参与下调DSPP基因表达。