Kliment Corrine R, Englert Judson M, Crum Lauren P, Oury Tim D
Department of Pathology, University of Pittsburgh School of Medicine Pittsburgh, PA, USA.
Int J Clin Exp Pathol. 2011 Apr;4(4):349-55. Epub 2011 Apr 12.
The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use.
8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay.
The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.
本研究的目的是开发一种改进的方法，用于评估纤维化肺中的胶原蛋白和蛋白质，同时减少动物使用量。
对8-10周龄的雄性C57BL/6小鼠进行一次气管内滴注青石棉或对照二氧化钛。在第14天收集肺组织，将整个肺干燥，或在CHAPS缓冲液中匀浆，用于羟脯氨酸分析。还使用改良的天狼星红比色96孔板分析法测定肺匀浆中不溶性和盐溶性胶原蛋白的含量。
羟脯氨酸分析显示，石棉处理小鼠的肺中胶原蛋白含量显著增加。在干燥的整个肺或整个肺匀浆中测定的胶原蛋白含量之间存在相同的结果。天狼星红板分析法显示肺匀浆中胶原蛋白含量显著增加，然而，该分析法严重高估了胶原蛋白的总量，并低估了对照肺和纤维化肺之间的变化。结论：所提出的方法能够准确量化整个肺中的胶原蛋白含量，并为从单个动物获取用于生化分析的额外匀浆样本提供了方法。天狼星红比色板分析法为测定肺胶原蛋白的相对变化提供了一种补充方法，但该方法倾向于高估通过金标准羟脯氨酸分析法获得的绝对值，并低估整体纤维化损伤。
