Gerstenfeld L C, Chipman S D, Glowacki J, Lian J B
Dev Biol. 1987 Jul;122(1):49-60. doi: 10.1016/0012-1606(87)90331-9.
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 approximately 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.(ABSTRACT TRUNCATED AT 400 WORDS)
本报告记录了体外成骨细胞的分化情况，表现为成骨细胞表型已知标志物的蛋白质增加了50至100倍。I型胶原蛋白和骨钙素的合成与积累、碱性磷酸酶活性以及基质钙化呈现出与体内骨骼发育过程中相似的时间关系。鸡胚成骨细胞祖细胞最初在基础培养基中低密度生长时被筛选出来。当以更高细胞密度传代培养到营养丰富的培养基中时，获得了近乎均匀的成骨细胞群体，碱性磷酸酶活性阳性细胞的富集率超过80%即可证明。对在有或没有10 mMβ-甘油磷酸酯（β-GPO4）（一种基质钙化的化学刺激物）存在的情况下培养的细胞随时间变化进行了比较。用β-GPO4处理的培养物在第12天培养单层汇合时出现可见的钙化。到第30天，可见大量大的钙化灶，钙（Ca）含量增加了20倍。相比之下，未处理的培养物钙含量仅增加了3倍，且钙化区域较小且分散。两种培养物中的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均无明显影响。两种培养物产生的主要胶原蛋白类型均为I型，通过溴化氰肽图谱分析和延迟还原分析未检测到III型。碱性磷酸酶活性在第18天迅速诱导增加约50倍，并在对照培养物中保持升高。然而，用β-GPO4处理的培养物在18天后酶活性迅速下降了80%。相比之下，骨钙素总水平在第18天诱导增加了100倍，并在整个检测时间段内，在对照培养物和用β-GPO4处理的培养物中均保持升高。虽然用β-GPO4处理的培养物和未处理的培养物中骨钙素的总体水平相同，但在用β-GPO4处理的培养物中，与矿化程度更高的基质相关的骨钙素多2至5倍。为了证实骨钙素与矿化区域的关联，通过将四环素活体标记与抗骨钙素和抗胶原蛋白抗体的免疫荧光染色相结合，对矿化与骨钙素和胶原蛋白进行了共定位。胶原蛋白和骨钙素在矿化区域均显示出强烈的定位。（摘要截断于400字）
