Ⅱ型胶原的研制及其在软骨细胞培养中的应用

Preparation Of Type Ⅱ Collagen and Use as Chondrocytes Culture Substratum

目的 :建立一套完善的提取、纯化、检测Ⅱ型胶原的生产系统 ,并以此产品应用在软骨细胞培养上。方法 :选取猪透明软骨 ,经盐酸胍、胃蛋白酶等多步抽提 ,以及DEAE纤维素的柱层析 ,制备出Ⅱ型胶原作为软骨细胞培养的基质。结果 :通过SDS PAGE电泳、氨基酸成份分析证实所制备的Ⅱ型胶原比Sigma(90 0 7 34 5 )的产品纯度高 ,以此Ⅱ型胶原为基质培养软骨细胞 ,原代至第 6代增殖快 ,6代以后的细胞呈成纤维状的去分化现象。结论 :自行研制的Ⅱ型胶原为高纯度的Ⅱ型胶原 ,应用在软骨细胞培养上 ,能使细胞增殖快 。

Objective: To establish a consummate system of extraction,purification and analysis of type Ⅱ Collagen that use as chondrocytes culture substratum.Methods:porcine cartilage extracted by guanidine HCl and pepsin,then,purificated by DAEA cellulose column,the type Ⅱ collagen was prepared.And that use as chondrocytes culture substratum.Results:The type Ⅱ collagen was purer than that produced by Sigma(9007 34 5) which examined by SDS PAGE electrophoresis,composition of amine acid testing.The chondrocytes that culture with this type Ⅱ collagen substratum was proved to be proliferated faster preceding six passages.Afterwards,the chondrocytes tended to assume fibroblastic dedifferentiation morphology.Conclusion:The type Ⅱ collagen that prepared had high purity.The chondrocytes that culture with it can keep the phenotype and viability for a long term and proliferated faster.