Puustjärvi K, Lammi M J, Kiviranta I, Helminen H J, Tammi M
University of Kuopio, Department of Anatomy, Finland.
Histochemistry. 1993 Jan;99(1):67-73. doi: 10.1007/BF00268023.
A rapid quantitation of proteoglycan synthesis distribution in intervertebral disc and endplates is described. Tissue blocks of disc (C7-Th1) in the midsagittal plane from ten female beagles were incubated in the presence of 35SO4 and prepared as histological slides. For comparison, sulphate incorporation rates in the C5-C6 discs were assayed by liquid scintillation. Autoradiographic film exposed against the labelled sections was developed and digitized for image analysis using a 256 grey level flat bed table scanner connected to a microcomputer. The film density versus dpm (disintegrations per minute) calibration was performed using a set of 35SO4-labelled glycosaminoglycan standards applied on the same film. Since section thickness, dpm calibration of the film density and the specific activity of sulphate in the medium were known, the incorporations per tissue volume could be calculated. The average incorporation rates of the anterior and posterior annulus fibrosus, nucleus pulposus and vertebral endplates were 5.2 +/- 0.9, 5.2 +/- 0.8, 4.5 +/- 0.6 and 4.1 +/- 0.8 pmol/mm3 per h (+/- SE, n = 10), respectively and closely corresponded to those obtained by liquid scintillation. This method offers a convenient and reproducible way to measure the rate of proteoglycan synthesis in large tissue sections but also in thin cartilaginous tissues such as the vertebral endplate.
本文描述了一种快速定量测定椎间盘和终板中蛋白聚糖合成分布的方法。从十只雌性比格犬的矢状面中间切取C7-Th1椎间盘组织块，置于35SO4存在的环境中孵育，然后制成组织学切片。作为对照，采用液体闪烁法测定C5-C6椎间盘的硫酸盐掺入率。将与标记切片相对放置的放射自显影片显影，并使用连接到微型计算机的256灰度级平板扫描仪进行数字化以进行图像分析。使用一组涂覆在同一张胶片上的35SO4标记的糖胺聚糖标准品对胶片密度与每分钟衰变数（dpm）进行校准。由于切片厚度、胶片密度的dpm校准以及培养基中硫酸盐的比活度已知，因此可以计算每组织体积的掺入量。纤维环前、后部分、髓核和椎体终板的平均掺入率分别为每小时5.2±0.9、5.2±0.8、4.5±0.6和4.1±0.8 pmol/mm3（±标准误，n = 10），与液体闪烁法测得的结果非常接近。该方法提供了一种方便且可重复的方式，不仅可以测量大组织切片中蛋白聚糖的合成速率，还能测量诸如椎体终板等薄软骨组织中的合成速率。
