摘要
[目的]通过对两种方法提取的骨胶原产量及特性的比较,推荐一种较为理想的提取皮质骨I型胶原的方法。[方法]以猪皮质骨为原料,去除软组织后劈成小骨块,经酒精梯度脱水、无水乙醚脱脂、盐酸脱钙、甲醇:氯仿(1:1,v/v)再脱脂处理后,骨块软化。然后将软化的骨块在高速粉碎机上粉碎成脱钙骨基质粉。分别采用传统的碱溶法和自建的增强酶解法对脱钙骨基质粉处理,经溶解、离心、透析、冻干等操作提取皮质骨胶原。最后对增强酶解法所得产物进行鉴定,并对两种提取方法的效率及得到的产物的外观、黏度、凝胶固化性进行比较。[结果]增强酶解法所得胶原的氨基酸组成、蛋白电泳、相对分子量大小、最大光吸收波长均符合I型胶原特征;和碱溶法比较,提取率由(57.8±4.96)%提高到(94.04±0.55)%;其相同浓度(0.03%,w/v)醋酸溶液黏度测定,增强酶解法为3.71,碱提法为2.81;在37.0℃,pH值7.35～7.45条件下行凝胶固化性检测,增强酶解法所提胶原凝胶液约10min可固化形成凝胶块,而碱提法所提胶原的溶液仍为黏性液体。[结论]增强酶解法提取的骨胶原是I型胶原;相对于碱提法,该法提取效率高,所得胶原纯度高、黏性好、固化性好,是一种较好的从猪皮质骨中制备I型胶原的方法。
[ Objective] To recommend an ideal method for extracting Ⅰ-collagen from cortical bone. [ Method ] The cortical bone of pig was splitted into small pieces after the soft tissues were cleaned up. The bone pieces were gradually dehydrated in alcohol, defatted in aether, decalcificated in hydrochloric acid, redefatted in chloroform： methanol （1：1, v/v） and became soft. The soft pieces were disintegrated into demineralized bone matrix （DBM） powder in a high speed mill, The osseins were extracted respectively by enhanced pepsin digestion method or alkall-solution method after the DBM powder was treated with desolving, centrifuging, dialyzing and lyophilization. The product got by enhanced pepsin digestion method was further confirmed. The extraction rate and appearance, viscosity and solidification of both products were analyzed and compared. [ Result ] The collagen produced by extraction of enhanced pepsin digestion method was confirmed to be Ⅰ-collagen by analyzing amino-acid composition, protein electrophoresis, relative moleculas weight and max wavelength about light absorption. The extraction rate of Ⅰ-collagen by enhanced pepsin digestion method was （94.0±14.96）% as comparision with by alkali-solution method （57.8±4.96）% and the viscosity test of acetic and solution at the concentration 0.03% （w/v） was 3.71 and 2.81 respectively. At the condition of 37°, pH 7.35 - 7.45, the collagen solution extracted by enhanced pepsin digestion method solidified and changed into a glue 10 minutes later, while the product solution by extraction with alkali-solution was still like a sticky liquid. [ Conclusion] Ossein extracted from cortical bone with enhanced pepsin digestion method is a real f-collagen. Compared with alkeli-solution extraction method, the enhanced pepsin digestion extraction method has advantages of higher extraction rate, better purity, better viscosity and fine solidification of the product, and is a choice to prepare the Ⅰ-collagen from cortical bone.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2007年第6期463-466,共4页
Orthopedic Journal of China
基金
国家863计划资助项目(2003AA205020)
关键词
皮质骨Ⅰ型胶原
增强酶解法
碱溶法
cortical bone
Ⅰ-collage
enhanced pepsin digestion method
alkali-solution method
