EXPRESSION OF DIFFERENTIATED FUNCTION BY MINERALIZING CULTURES OF CHICKEN OSTEOBLASTS
EXPRESSION OF DIFFERENTIATED FUNCTION BY MINERALIZING CULTURES OF CHICKEN OSTEOBLASTS
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DOI：
10.1016/0012-1606(87)90331-9
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发表时间：
1987-07-01
影响因子：
2.7
通讯作者：
LIAN, JB
中科院分区：
文献类型：
--
作者：
GERSTENFELD, LC;CHIPMAN, SD;LIAN, JB
该报告记录了成骨细胞的体外分化，蛋白质增加了 50-100 倍，这些蛋白质是成骨细胞表型的已知标志物。 I 型胶原蛋白和骨钙素的合成和积累、碱性磷酸酶活性和基质钙化显示出相似的时间关系，类似于体内骨骼发育过程中所见的时间关系。通过在基本培养基中以低密度进行初始生长来选择鸡胚胎成骨细胞祖细胞。 在以较高细胞密度传代到营养丰富的培养基中后，获得了接近均质的成骨细胞群体，碱性磷酸酶活性阳性细胞富集超过80%就证明了这一点。对在存在或不存在 10 mM β-磷酸甘油 (β-GPO4)（一种基质钙化的化学刺激剂）的情况下生长的细胞随时间的变化进行了比较。用β-GPO4处理的培养物在第12天当培养物单层变得汇合时显示出可见的钙化。到第 30 天，可以看到许多大的钙化灶，并且观察到钙 (Ca) 含量增加了 20 倍。相比之下，未经处理的培养物的钙含量仅增加了 3 倍，并且有许多较小的弥漫性钙化区域。两种培养物之间的DNA、RNA和总蛋白水平几乎相同，表明β-GPO4对细胞增殖或转录活性没有显着影响。两种培养物产生的主要胶原蛋白类型是 I 型，根据 CNBr 肽图谱和延迟还原分析确定，没有检测到 III 型胶原蛋白。碱性磷酸酶活性显示出快速的。第 18 天诱导了 50 倍，并且在对照培养物中仍保持较高水平。然而，用β-GPO4处理的培养物显示18天后酶活性迅速下降80%。相反，总骨钙素水平在第18天显示诱导100倍，并且在整个检查的时间段内对照和β-GPO4处理的培养物中保持升高。虽然骨钙素的总体水平在β-GPO4处理和未处理的培养物中相同，但2至5倍的骨钙素与β-GPO4处理的培养物的矿化程度更高的基质相关。为了确认骨钙素与矿化区域的关联，通过将四环素活体标记和抗骨钙素和抗胶原抗体的免疫荧光染色相结合，进行矿物质与骨钙素和胶原蛋白的共定位。胶原蛋白和骨钙蛋白都在矿化区域表现出强烈的定位。该培养系统定义了分化过程中成骨细胞功能的特定标志物的表达及其与基质钙化的关系。因此，这些成骨细胞培养物提供了一个独特的模型，用于研究成骨细胞分化和基质钙化过程中骨特异性蛋白和基因的调节。
This report documents osteoblast differentiation in vitro, as demonstrated by the 50-100x increase of proteins which are known markers of the osteoblast phenotype. Collagen type I and osteocalcin synthesis and accumulation, alkaline phosphatase activity, and matrix calcification show similar temporal relationships that are analogous to those seen during in vivo bone development. Chicken embryonic osteoblast progenitor cells were selected by initial growth at low densities in minimal medium. Upon subcultivation into nutrient-enriched medium at higher cell densities, near homogeneous populations of osteoblasts were obtained as demonstrated by the greater than 80% enrichment of cells positive for alkaline phosphatase activity. A comparison was made between cells grown in the presence or absence of 10 mM .beta.-glycerolphosphate (.beta.-GPO4), a chemical stimulant of matrix calcification, as a function of time. Cultures treated with .beta.-GPO4 showed visible calcification at Day 12 when culture monolayers became confluent. By Day 30, numerous large foci of calcification were visible and a 20-fold increase in calcium (Ca) content was observed. In contrast, untreated cultures had only a 3-fold increase in Ca content with many smaller diffuse areas of calcification. DNA RNA, and total protein levels were nearly identical between the two cultures, indicating that .beta.-GPO4 had no marked effect on either cell proliferation or transcriptional activity. The major collagen type produced by either culture was type I, with no detectable type III as determined by CNBr peptide mapping and delayed reduction analysis. Alkaline phosphatase activity showed a rapid .apprx. 50-fold induction by Day 18 and remained elevated in control cultures. However, cultures treated with .beta.-GPO4 demonstrated a rapid 80% decline of enzyme activity after 18 days. In contrast, total osteocalcin levels showed a 100-fold induction by Day 18 and remained elevated in both control and .beta.-GPO4-treated cultures throughout the time period examined. While the overall levels of osteocalcin were the same in .beta.-GPO4-treated and untreated cultures, 2- to 5-fold more osteocalcin was associated with the more mineralized matrices of the .beta.-GPO4-treated cultures. In order to confirm the association of osteocalcin with areas of mineralization, co-localization of mineral to osteocalcin and collagen was carried out by combining vital labeling with tetracycline and immunofluorescent staining with anti-osteocalcin and anti-collagen antibodies. Both collagen and osteocalcin showed strong localization with areas of mineralization. This culture system defined the expression of specific markers of osteoblast function during differentiation and their relationships to matrix calcification. Thus, these osteoblast cultures provide a unique model in which to study the regulation of bone-specific proteins and genes during osteoblast differentiation and matrix calcification.