乳铁蛋白胃肠道降解产物促骨生长功能研究及其活性中心片段的确定

Lactoferrin is a newly discovered function of substance with osteogenic activity from the milk, non-toxic, harmless. The laboratory confirms the improvement of oral lactoferrin on ovariectomized rat model of osteoporosis through years of research. However, complex mechanism of action and its degradation of the active center of fragments are yet to be clarified.In this study, bovine lactoferrin (bLF) will be hydrolyzated by pepsin and pancreatin or simulating the human digestion environment to prepare the products of lactoferrin enzymic hydrolysate. BrdU-ELISA are evaluated the digestive enzyme hydrolysis of lactoferrin at different times after the degradation fragment of the proliferative activity of osteoblasts, and filter out most of the osteogenic activity component. By gel chromatography and reversed phase chromatography, the hydrolysates will be further separated, and than the significantly osteogenesis activity peptides are screened out by BrdU-ELISA. To get the primary sequence information of active centre component of lactoferrin are identified by HPLC-MS and protein N-terminal sequencing, and than the osteogenic active center petide was obtained by Solid-phase synthesis.The proliferation rate of osteoblasts and the alkalescent phosphoric acid enzymatic expression of osteoblast marker at last will be used to appraise the osteogenesis function of the lactoferrin osteoblast activity peptide.In this study the osteogenic active center peptide will be clarified finally.

乳铁蛋白是乳中最新发现具有明显促进骨生长的功能因子。本课题组充分证实了口服乳铁蛋白对大鼠骨质疏松症的良好改善作用。然而经口服的乳铁蛋白在体内复杂酶体系作用下会发生降解，其降解产物仍然具有成骨活性，而其发挥成骨活性的中心片段尚不清楚。本研究拟以牛乳铁蛋白为原料，分别制备模拟胃部、肠道及胃肠道共同作用共3种条件下，不同时间处理后乳铁蛋白的降解产物，采用5-溴脱氧尿嘧(BrdU-ELISA)法评价乳铁蛋白降解产物体外对成骨细胞成骨活性的影响；采用凝胶层析色谱、RP-HPLC等方法对其酶解产物活性部分进行分离纯化，HPLC-MS和蛋白质N端测序等手段对所得活性肽段进行组成鉴定，获取具有成骨活性肽段一级序列的精确信息，运用固相合成法合成具有成骨活性相应肽段，通过细胞增殖率及成骨细胞成骨标志物碱性磷酸酶表达量的测定，评价具有成骨活性多肽的成骨功能；确定乳铁蛋白促进成骨功能的活性中心肽端的位置。

本研究以牛乳铁蛋白为原料，模拟胃肠道消化环境分别制备了经胃蛋白酶和胰酶以及先经胃蛋白酶后经胰酶作用的乳铁蛋白酶解产物；采用SDS-PAGE电泳方法监测了乳铁蛋白降解产物分子量分布的变化；BrdU-ELISA法评价了乳铁蛋白降解产物对成骨细胞增殖活性的影响；凝胶层析及反相层析法对酶解产物进一步分离纯化，BrdU-ELISA法筛选出具有成骨活性的单一组分，并以质谱、氨基酸测序等手段对所得活性组分进行了组成的鉴定，同时对该活性组分固相分段合成，经BrdU-ELISA法筛选，确定了乳铁蛋白促进成骨的活性肽段的位置。本研究对明确乳铁蛋白体内降解产物的成骨功能，阐明口服乳铁蛋白的体内成骨作用机理具有重要意义。