氢氧化钙对人牙髓细胞中骨涎蛋白基因转录的影响

Calcium hydroxide regulates bone sialoprotein gene transcription in human dental pulp cells

目的观察氢氧化钙对人牙髓细胞中骨涎蛋白基因转录的影响，研究氢氧化钙诱导矿化的机制，为研制更有效的盖髓剂提供实验依据。方法收集天津医科大学口腔医院口腔颌面外科门诊因正畸拔除的双尖牙15颗，提取牙髓组织并进行细胞培养，将培养传代的人牙髓细胞分成不同浓度（0．012、0．120、0．400和1．200mmol／L）氢氧化钙实验组和对照组，提取总RNA，通过实时定量聚合酶链反应实验，检测确定人牙髓细胞中氢氧化钙对骨涎蛋白mRNA水平的最佳刺激浓度。同时，检测最佳浓度氢氧化钙在不同时间点（0、3、6、12和24h）对骨涎蛋白、核心结合蛋白因子2（runt-relatedtranscriptionfactor-2，Runx-2）和成骨细胞特异性转录因子（osterix，OSX）mRNA水平的影响；通过瞬时转染实验，将人骨涎蛋白基因不同长度片段的启动子插入荧光素酶基因中，通过脂质体瞬时转染进人牙髓细胞，检测骨涎蛋白启动子在氢氧化钙实验组和对照组中转录活性的变化。组内和组间比较均采用两独立样本t检验，以双侧P〈0．05为差异有统计学意义。结果实时定量聚合酶链反应实验中，最佳刺激浓度1．200mmol／L氢氧化钙作用人牙髓细胞12h后，骨涎蛋白mRNA的水平增长为（2．86±0．09），而1．200mmol／L氢氧化钙在不同的时间点（0、3、6、12和24h）刺激细胞，骨涎蛋白mRNA水平6h时为（1．45±0．36），12h时增长到（2．66±0．18）；Runx-2mRNA水平在6h时为（2．38±0．08），12h时增长到（2．73±0．16），24h时下降至初始水平；OSXmRNA的水平从12h开始增长，24h达峰值（3．30±0．06）。瞬时转染实验也证实了氢氧化钙刺激人牙髓细胞12h后，人骨涎蛋白基因-84LUC-868LUC启动子转录活性氢氧化钙刺激组比对照组增强了2．00—2．60倍。结论氢氧化钙诱导矿化中增强了人骨涎蛋白基因的转录，转录位点可能位于人骨涎蛋白基因启动子的-84～-868区域。

Objective To analyze the effects of calcium hydroxide [ Ca（ OH ）21 on transcription of the bone sialoprotein（BSP） gene in human dental pulp cells. Methods Human dental pulp tissues were collected from extracted teeth for orthodontic reason. In cell culture media, different dose （0. 012,0. 120, 0. 400 and 1. 200 mmol/L）of Ca（OH）2 was added. Total RNA of cells were extracted. The best dose of Ca （OH） 2 on human BSP was determined with the real-time polymerase chain reaction （PCR）. Further, the time（0,3,6,12,24 h） effects of the best dose Ca（OH） 2 on human BSP, runt-related transcription factor-2 （Runx-2） and osterix（OSX） mRNA levels were determined with PCR. Further method included transient transfection assays, linking chimeric constructs of the human BSP gene promoter to a luciferase reporter gene, then ransfected using lipofectamine in cells and measured the luciferase activities of BSP gene promoter. Results With the real-time PCR, the optimal Ca （OH）2 concentration was determined as 1. 200 mmol/L With this concentration at different time points（0, 3, 6, 12 and 24 h）, the levels of BSP mRNA increased at 6 h （ 1.45 ±0. 36） , reached maximal at 12 h（2. 66 ±0. 18） ;the levels of Runx-2mRNA increased at 6 h（2. 38 ± 0. 08）, at 12 h （2. 73 ± 0. 16）, and decreased at 24 h. OSX mRNA could be recognized at 12 h, reached maximal levels at 24 h（3.30 ± 0. 062）. Transient transfection assays showed that treatment of human dental pulp cells with Ca（OH）2 （1. 200 mmol/L） increased the luciferase activities of the constructs between -84LUC and -868LUC at 12 h（2.00 -2. 60 fold）. Conclusions This study demonstrate that Ca（OH） = could stimulate BSP transcription between -84LUC and -868LUC in the human BSP gene promoter in human dental pulp cells.