Cooney Timothy E, Konieczko Elisa M, Roach Lindsey, Poole Bradley
Orthopedic Research, Hamot Medical Center, 201 State St, Erie, PA 16550, USA.
Orthopedics. 2009 Feb;32(2):95.
Studies have intimated a role for endogenous opioids in skeletal ontogeny. We hypothesized that this role may be confined to the perinatal period. We, therefore, examined both fetal and postnatal rat long bones to determine the pattern, if any, of mu receptor expression. Perfused long bones and brains were harvested from 3 to 4 each of near-term fetuses, 3- to 4-week-old neonates, and 6-week-old juveniles. Tissues were decalcified, embedded, sectioned, quenched of peroxidase, and then blocked. Sections were incubated overnight with either rabbit antirat mu receptor IgG or naïve rabbit IgG (control) at 4 degrees C. The next day, sections were washed, blocked again, and incubated with biotin-labeled secondary antibody, streptavidin-peroxidase conjugate, and a chromogen substrate with intervening wash steps. Slides were counterstained with hematoxylin and coverslipped. Digital photomicrographs were then imported into an image analysis program and the percent area of stained cortical and trabecular bone quantified. Mu receptor expression decreased significantly with age. Approximately 25% of the area of fetal long bones stained positively, including the endosteum, periosteum, and the growth plate. Little or no nonspecific staining occurred. Staining in neonatal tissue was diminished to <11% of the area and involved areas of apparent remodeling; chondrocytes in the growth plates failed to stain. Finally, juvenile bone evidenced staining approaching background levels produced by control slides (approximately 2%). Mu receptors are abundant in developing rat long bones during fetal development but become progressively less abundant postnatally. This infers a role for endogenous opioids during skeletal ontogeny.
研究表明内源性阿片类物质在骨骼发育过程中发挥作用。我们推测这种作用可能局限于围产期。因此，我们检查了胎儿和出生后大鼠的长骨，以确定μ受体表达的模式（如果存在的话）。从3至4只近足月胎儿、3至4周龄新生儿和6周龄幼鼠中分别采集灌注后的长骨和大脑。组织经脱钙、包埋、切片、过氧化物酶淬灭，然后进行封闭。切片在4℃下与兔抗大鼠μ受体IgG或单纯兔IgG（对照）孵育过夜。第二天，切片经洗涤、再次封闭，然后与生物素标记的二抗、链霉亲和素 - 过氧化物酶结合物以及显色底物孵育，并进行中间洗涤步骤。玻片用苏木精复染并加盖玻片。然后将数码显微照片导入图像分析程序，对染色的皮质骨和小梁骨的面积百分比进行量化。μ受体表达随年龄显著下降。胎儿长骨约25%的面积呈阳性染色，包括骨内膜、骨膜和生长板。几乎没有非特异性染色。新生儿组织中的染色减少至面积的<11%，且涉及明显重塑的区域；生长板中的软骨细胞未染色。最后，幼鼠骨骼的染色接近对照玻片产生的背景水平（约2%）。μ受体在胎儿发育期间的大鼠长骨中丰富，但出生后逐渐减少。这暗示了内源性阿片类物质在骨骼发育过程中的作用。
