Wang Yu-Hsiung, Liu Yaling, Buhl Kathy, Rowe David W
Department of Pediatric Dentistry, School of Dental Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-1610, USA.
J Bone Miner Res. 2005 Jan;20(1):5-14. doi: 10.1359/JBMR.041016. Epub 2004 Oct 25.
Primary calvarial osteoblast cultures derived from type I collagen promoter-GFP reporter transgenic mice were used to examine progression of the osteoblast lineage. This system was validated by assessing the effect of PTH on osteoblast growth in real time. The anabolic effect of PTH seemed to be the result of enhanced osteoblast differentiation rather than expansion of a progenitor population.
Activation of green fluorescent protein (GFP) marker genes driven by Col1a1 promoter fragments has been associated with the level of osteoblast differentiation. GFP-marked cultures provide an approach to continuously monitor the level of osteoblast differentiation in real time without the termination of cultures.
Neonatal calvarial cells transgenic for pOBCol2.3GFP and pOBCol3.6GFP were used to establish calvarial osteoblast cultures. Parathyroid hormone (PTH) was added either continuous (days 1-21) or transient (days 1-7) to examine its diverse effect on osteoblast differentiation in cultures for 21 days. Three fluorescent markers were used: (1) pOBCol3.6GFP, which is activated in preosteoblastic cells; (2) pOBCol2.3GFP, which is restricted to differentiated osteoblasts; and (3) xylenol orange (XO), which stains the mineralized nodules. Progression of osteoblast differentiation indicated by fluorescent markers was documented throughout the entire period of culture. Recorded fluorescent images were analyzed in the patterns of expression and quantitated in the area of expression.
Continuous PTH blocked osteoblast differentiation, which was evident by the attenuation of pOBCol3.6GFP and an absence of pOBCol2.3GFP. In contrast, transient PTH inhibited the initial osteoblast differentiation but ultimately resulted in a culture with more mineralized nodules and enhanced osteoblast differentiation expressing strong levels of pOBCol3.6GFP and pOBCol2.3GFP. Quantitative analysis showed that transient PTH first decreased then later increased areas of GFP expression and XO staining, which correlated with results of Northern blot and alkaline phosphatase activity. Transient PTH caused a decrease in DNA content during the treatment and after the removal of PTH.
GFP-marked cultures combined with fluorescent image analysis have the advantage to assess the effect of PTH on osteoblast differentiation in real time. Results suggest that the anabolic effect of transient PTH is caused by an enhancement in osteoblast differentiation rather than an increase in the population of progenitor cells.
使用源自I型胶原启动子 - GFP报告基因转基因小鼠的原代颅骨成骨细胞培养物来研究成骨细胞谱系的进展。通过实时评估甲状旁腺激素（PTH）对成骨细胞生长的影响来验证该系统。PTH的合成代谢作用似乎是成骨细胞分化增强的结果，而不是祖细胞群体的扩增。
由Col1a1启动子片段驱动的绿色荧光蛋白（GFP）标记基因的激活与成骨细胞分化水平相关。GFP标记的培养物提供了一种在不终止培养的情况下实时连续监测成骨细胞分化水平的方法。
使用转染pOBCol2.3GFP和pOBCol3.6GFP的新生颅骨细胞建立颅骨成骨细胞培养物。连续（第1 - 21天）或短暂（第1 - 7天）添加甲状旁腺激素（PTH），以研究其对培养21天的成骨细胞分化的不同影响。使用了三种荧光标记物：（1）pOBCol3.6GFP，在成骨前体细胞中被激活；（2）pOBCol2.3GFP，仅限于分化的成骨细胞；（3）二甲酚橙（XO），用于染色矿化结节。在整个培养期间记录由荧光标记物指示的成骨细胞分化进展。对记录的荧光图像进行表达模式分析并在表达区域进行定量。
连续添加PTH会阻断成骨细胞分化，这在pOBCol3.6GFP减弱和pOBCol2.3GFP缺失中很明显。相比之下，短暂添加PTH会抑制初始成骨细胞分化，但最终导致培养物中有更多矿化结节和成骨细胞分化增强，表达高水平的pOBCol3.6GFP和pOBCol2.3GFP。定量分析表明，短暂添加PTH首先会降低然后增加GFP表达和XO染色的面积，这与Northern印迹和碱性磷酸酶活性的结果相关。短暂添加PTH在处理期间和去除PTH后会导致DNA含量降低。
GFP标记的培养物与荧光图像分析相结合具有实时评估PTH对成骨细胞分化影响的优势。结果表明，短暂添加PTH的合成代谢作用是由成骨细胞分化增强引起的，而不是祖细胞群体的增加。
