胰蛋白酶及Ⅱ型胶原酶消化获取关节软骨细胞

Harvest of articular cartilage cells using digestion of trypsin and type Ⅱ collagenase

背景:近年来,众多研究欲采用体外分离培养的关节软骨细胞作为修复缺损的关节软骨的种子细胞,然而,获得纯化的具有生物活性的关节软骨细胞较为困难。目的:拟运用胰蛋白酶与Ⅱ型胶原酶联合消化获取关节软骨细胞。方法:从SD大鼠的正常股骨及胫骨关节表面获取关节软骨,先后运用0.25%的胰蛋白酶和0.2%Ⅱ型胶原酶消化,显微镜下见大量细胞游离后,弃去大块的未消化的关节软骨碎片,离心,去上清,PBS洗涤2次后,加入软骨细胞原代培养液进行培养、增殖。应用甲苯胺蓝及苏木精-伊红染色方法检验所得细胞是否为关节软骨细胞。结果与结论:在严格掌握酶的浓度及消化时间的前提下,通过0.25%胰蛋白酶和0.2%Ⅱ型胶原酶联合酶解关节软骨的方法,成功从大鼠股骨及胫骨关节软骨内分离培养出细胞,并经过甲苯胺蓝染色及苏木精-伊红染色证实,所得的细胞为具有生物活性的关节软骨细胞。

BACKGROUND：Recent years,most studies have attempted to repair defected articular cartilage using isolated articular cartilage cells.However,it is difficult to obtain purified articular cartilage cells that have biological activity.OBJECTIVE：To harvest articular cartilage cells using combined digestion of trypsin and type Ⅱ collagen.METHODS：Articular cartilages were obtained from the surface of normal femoral head of SD rats,digested by trypsin（0.25%） and collagease Ⅱ（0.2%）.After observing numerous articular cartilage cells free under a phase contrast microscope,the big pieces of articular cartilage was abandoned,followed by centrifugation and washing with phosphate buffered saline for 2 times.And then,the mixture was cultured in cartilage cells culture medium.Toluidine blue and hematoxylin-eosin staining were used to identify the articular cartilage cells.RESULTS AND CONCLUSION：With seriously controlling the concentrate of enzyme and the time of digestion,through enzymolysis of articular cartilage cells by trypsin（0.25%） and type Ⅱ collagease（0.2%）.Articular cartilage cells were obtained by isolating and cultivating from femoral shaft of SD rats,which was identified with toluidine blue and hematoxylin-eosin staining.