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

与衰老相关的骨质流失的一个重要原因可能是骨质形成的减少。我们利用有限稀释分析和连续传代培养实验，对老龄（24周龄）和幼龄（6周龄）雌性Wistar大鼠的骨祖细胞数量、增殖和分化能力以及自我更新能力进行了表征和比较。从腰椎（L1-L6）外植体中获得细胞，在α-微量必需培养基（α-MEM）、10%胎牛血清和50mg/ml抗坏血酸中培养，添加或不添加地塞米松（Dex）或孕酮（Prog）。两组细胞群体的生长曲线相似，老龄动物和幼龄动物的群体倍增时间分别为28.3和27.5 h。两个年龄组的骨祖细胞在Dex或Prog的存在下培养时形成骨结节。在10 nM Dex的存在下进行极限稀释分析，结果显示老年大鼠和幼龄大鼠的成骨细胞数量无显著差异，成纤维细胞（CFU-F）、成纤维细胞（AP+CFU-F）和成骨细胞（CFU-O）的集落形成单位数量较多。在3mm Prog存在下的限制性稀释分析显示，老年组和青年组之间CFU-F、AP+CFU-F或CFU-O的数量没有差异。通过连续传代、极限稀释分析和菌落测定，确定了多达6次传代培养中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传代细胞中对dex有反应的骨祖细胞的总数比第一次传代细胞显著增加，但这些祖细胞的频率没有显著增加。从第三次传代开始，骨祖细胞的频率呈线性下降，六次传代后未观察到任何骨祖细胞。在含prog的培养基中也得到了类似的结果。结果表明，老龄大鼠和幼龄大鼠椎体骨细胞群的细胞倍率、生长特征、CFU-F和成骨细胞数量相似。这表明与衰老相关的骨质流失不是由骨祖细胞数量减少引起的。然而，老年大鼠的细胞群在体外显示出自我更新能力的降低，这最终会转化为成骨细胞数量的减少，这可能是老年动物骨形成减少的部分原因。先前的实验表明，来自成年雌性大鼠腰椎骨外植体的骨细胞群含有骨祖细胞，这些骨祖细胞需要Dex或Prog增殖并分化为完全分化的成骨细胞。我们已经证明，在性成熟后的雄性大鼠中无法检测到prog依赖性群体，但在两性青春期前的大鼠中都存在，并且可以通过将外植体培养在含有17 β-雌二醇（10^<-9>-10^<-8 >m）的培养基中诱导成年雄性衍生群体。这表明，成年雌性衍生种群中依赖于Frog和Dex的骨祖细胞可能是不同的种群，并且依赖于prog的骨祖细胞的存活和/或其增殖能力依赖于雌激素的存在。结果表明，Prog通过调节对Prog有反应的骨祖细胞的种类，在维持骨量中起重要作用。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