组织细胞中胶原蛋白的提取及ECL-Western blot分析

Extraction and ECL-Western blot analysis of collagen typesⅠ and Ⅱ in tissues and cells

目的建立一种可简便、有效的胶原蛋白提取、纯化及型别分析的测定方法。方法用破碎、酶解和盐析3步法提纯胶原,用ECL-Westernblot测定细胞基质胶原(200例)、正常组织及工程化修复组织中的胶原(30例)。结果该法提取胶原的时间周期短,得率高于传统方法的2.5倍,纯度达到了PAGE纯化的水平。结论该法具有简便、快速、灵敏和无放射性污染等优点,为组织工程胶原的深入研究提供了有价值的测试手段。

Aim To establish a simple and effective assay used to extract, purify and type collagen. Methods Collagen types Ⅰ and Ⅱ were extracted by use of three steps: crushing, acidolysing and salting out. Contents of collagen Ⅰ and Ⅱ extracted from cell matrix, normal and engineering tissues were detected by Western blot. Results The established method could shorten extracted duration, the output rate was 2.5 times higher than that from traditional extraction method, and the obtained collagens were proved to have high electrophoretic purity. Conclusion a simple, rapid, sensitive and radioactive uncontaminated method is established successfully. The method provide a valuable detection means for deeply studying tissue engineering collagen.