成骨不全病人原代成骨细胞中胶原蛋白提取方法改良

摘要

目的：本文对成骨不全患者骨骼中胶原蛋白的提取方法进行探讨，拟建立较为稳定的和可控的提取技术，为该病的治疗寻找新思路。方法：选取成骨不全患者在接受外科矫形手术中废弃的骨骼组织，经原代细胞培养后，采用盐析法配合胃蛋白酶消化提取细胞内和培养液中胶原蛋白。分别选取0.05~0.2mM抗坏血酸、0.1~0.25mMβ-氨基丙腈（BAPN）、0.5~1.5mg/mL胃蛋白酶进行梯度实验，比较胶原蛋白提取效果。抗坏血酸作用时间2到5天，每间隔24小时取样一次。胃蛋白酶作用时间，分别为5小时、10小时和20小时三个时间点进行比对。结果：从细胞中和培养液中提取的胶原蛋白，最适的胃蛋白酶消化时间为5小时，作用浓度1mg/mL。培养液中抗坏血酸浓度在0.1~0.15mM之间，β-氨基丙腈浓度达到0.1mM时，连续培养96h以上胶原蛋白产出量较高。结论：实验初步说明，通过优化后的胶原蛋白提取方法，可有效提取患者原代成骨细胞及细胞培养液中的胶原蛋白，有助于成骨不全相关研究的继续深入。%Objective:The paper aims to establish a stable and controllable method for collagen extraction from primary osteoblasts derived from osteogenesis imperfecta bone tissues.Methods:The primary osteoblasts were selected from the bone tissue of osteogenesis imperfecta patients who received deformity treatments. Collagen secreted from the cellular layer and culture medium was extracted by salting-out method and salting-out combined with pepsin digestion method, respectively. The different concentrations of L-ascorbate,ß-amino-propio-nitrille (BAPN) and pepsin were compared between 0.05~0.2mM, 0.1~0.25mM and 0.5~1.5mg/mL respectively. Different time points for L-ascorbate treatment were set at 2 days to 5 days, with an interval of 24 hours. For the pepsin digestion treatment, 5h, 10h and 20h time points were compared.Results: For the collagen extraction from both primary osteoblast cells and its cellular medium, the optimal pepsin digestion concentration was 1mg/mL with the treatment of 5 hours. High amount of collagen extracted from cellular medium of primary osteoblast cells was produced with the combination treatment of 0.1~0.15mM L-ascorbate and 0.1mM BAPN for 96 hour.Conclusions: The results indicated that collagen in primary osteoblast and culture medium can be extracted effectively by the optimized method. And it will promote in-depth study on Osteogenesis imperfecta.