Progesterone stimulates osteoprogenitor proliferation and differentiation in osteoblast-like cell populations derived from adult female rat vertebrae

与衰老相关的骨丢失的一个重要原因可能是骨形成的下降。我们的特点和比较的数量，增殖和分化能力和自我更新能力的老年（24周龄）和年轻（6周龄）的雌性Wistar大鼠骨祖细胞的有限稀释分析和连续传代实验。从腰椎（L1-L 6）外植体的生长物获得细胞，并在α-最小必需培养基（α-MEM）、10%FBS和50 mg/ml抗坏血酸（含或不含地塞米松（Dex）或孕酮（Prog））中生长。两个年龄组细胞群的生长曲线相似，老年和年轻动物的群体倍增时间分别为28.3和27.5 h。当在Dex或Prog存在下培养时，来自两个年龄组的骨祖细胞形成骨结节。在10 nM Dex存在下的有限稀释分析显示，老年大鼠和年轻大鼠在N-LDL水平上没有差异。 ...更多信息 集落形成单位-成纤维细胞（CFU-F）、碱性磷酸酶阳性集落形成单位-成纤维细胞（AP+CFU-F）或集落形成单位-成骨细胞（CFU-O）的数量。在3 mM Prog存在下的有限稀释分析显示，老年组和年轻组之间的CFU-F、AP+CFU-F或CFU-O的数量没有差异。使用连续传代培养、有限稀释分析和集落测定，确定了多达6次传代培养的两种祖细胞类型的频率和数量。在含Dex的培养基中，继代培养导致第二次继代培养细胞中Dex反应性骨祖细胞的总数比第一次继代培养细胞显著增加，而这些祖细胞的频率没有显著增加。从第三次继代开始，骨祖细胞的频率呈线性下降，六次继代后均未观察到骨祖细胞。在含Prog-containing培养基中获得了类似的结果。结果表明，老年大鼠和青年大鼠的椎骨骨细胞群的细胞群体倍增时间、生长特性、CFU-F和骨祖细胞的数量相似。这表明与衰老相关的骨丢失不是由骨祖细胞数量减少引起的。然而，来自老年大鼠的细胞群在体外显示出自我更新能力降低，以前的实验已经证明，来自成年雌性大鼠腰椎骨外植体的骨细胞群含有需要Dex或Prog增殖和分化的骨祖细胞，完全分化成骨细胞我们已经证明，在性成熟后的雄性大鼠中不能检测到Prog-dependent群体，但在青春期前的两种性别大鼠中都存在，并且可以通过在含有17 β-雌二醇（10^-10 ^M）的培养基中培养外植体来诱导成年雄性衍生群体<-9><-8>。这表明成年雌性衍生群体中的Frog和Dex依赖性骨祖细胞可能是不同的群体，并且Prog-dependent骨祖细胞的存活和/或其增殖能力取决于雌激素的存在。结果提示，Prog通过调节对Prog应答的骨祖细胞类别，在维持骨量方面起重要作用。少

A significant contribution to the bone loss associated with aging is likely to be a decline in bone formation. We have characterized and compared the number, capacity for proliferation and differentiation and the self-renewal ability of osteoprogenitors of aged (24-week-old) and young (6-week-old) female Wistar rats using limiting dilution analyses and continuous subculture experiments. Cells were obtained from outgrowths of explants of lumbar vertebrae (L1-L6) and grown in α-minimal essential medium (α-MEM), 10% FBS and 50mg/ml ascorbic acid with or without dexamethasone (Dex) or progesterone (Prog). Growth curves for cell populations of both age groups were similar with population doubling times of 28.3 and 27.5 h for the aged and young animals, respectively. Osteoprogenitors from both age groups formed bone nodules when cultured in the presence of either Dex or Prog. Limiting dilution analysis in the presence of 10 nM Dex showed no difference between the aged and young rats in the n … More umber of colony forming units-fibroblast (CFU-F), alkaline phosphatase-positive colony forming units-fibroblast (AP+CFU-F) or colony forming units-osteoblast (CFU-O). Limiting dilution analysis in the presence of 3 mM Prog showed no differences in the numbers of CFU-F, AP+CFU-F or CFU-O between the aged and young groups. Frequencies and numbers of both progenitor types were determined for up to six subcultures using continuous subculturing, limiting dilution analysis, and colony assays. In Dex-containing medium, subculturing resulted in a significant increase in the total number of Dex-responsive osteoprogenitors in second subculture cells over first subculture cells without a significant increase in the frequency of these progenitors. From the third subculture onward, the frequency of osteoprogenitors decreased in a linear manner and none were observed after six subcultures. Similar results were obtained in Prog-containing medium. Results indicate that the cell population doubling times, growth characteristics, and the number of CFU-F and osteoprogenitors in vertebral bone cell populations from aged rats and young rats are similar. This suggests that the bone loss associated with aging is not caused by a decrease in osteoprogenitor cell number. However, cell populations from the aged rats showed a reduced capacity for self-renewal in vitro, which would ultimately translate into a reduced number of osteoblasts and might be partly responsible for a decrease in bone formation in aged animals.Previous experiments have demonstrated that bone cell populations derived from explants of lumbar vertebral bone of adult female rats contain osteoprogenitors that require Dex or Prog to proliferate and differentiate into fully differentiated bone-forming osteoblasts. We have shown that the Prog-dependent population can not be detected in male rats after sexual maturation but is present in prepubertal rats of both sexes, and can be induced in adult male-derived populations by culturing the explants in media containing 17 β-estradiol (10^<-9>-10^<-8>M). This suggested that the Frog- and Dex- dependent osteoprogenitors in adult female-derived populations were likely to be distinct populations and that the survival of the Prog-dependent osteoprogenitors and/or their ability to proliferate is dependent on the presence of estrogen. The results imply that Prog plays an important role in maintaining bone mass through regulating the class of osteoprogenitors responsive to Prog. Less