原代培养差速贴壁法分离纯化大鼠腰椎间盘髓核细胞

Primary culture and purification of rat nucleus pulposus cells by anchorage velocity-dependent separation method

背景:髓核细胞在体外培养过程中存在表型丢失问题,包括Ⅱ型胶原、aggrecan、Sox-9等表达的下降,由类软骨细胞向类纤维样细胞转化。目的:体外原代培养差速贴壁法分离纯化大鼠腰椎间盘髓核细胞。方法:经胰蛋白酶、Ⅱ型胶原酶先后消化Wistar大鼠椎间盘髓核组织,第1,2代细胞传代时采用差速贴壁法分离纯化髓核细胞。结果与结论:分离纯化后的第3代髓核细胞呈圆形或多角形,活力强,苏木精-伊红染色细胞核染成均一蓝黑色,胞浆呈现淡粉色;Ⅱ型胶原免疫组织化学染色阳性细胞比例为97%;aggrecan免疫组织化学染色阳性细胞比例为95%;扫描电镜可见细胞内有高尔基体、粗面内质网和游离核糖体,未见线粒体,可见少量板层小体;CCK-8生长曲线显示细胞经过2d的生长潜伏期,3d的指数生长期,进入生长停滞期。说明原代培养、两次差速贴壁法分离纯化的大鼠髓核细胞代谢旺盛、表型一致。

BACKGROUND：Nucleus pulposus cells have the problem of phenotype loss during in vitro culture,such as the decreasing expression of type II collagen,Aggrecan and Sox-9,leading to conversion of cartilage-like cells to fibroblast-like cells.OBJECTIVE：To primarily culture rat nucleus pulposus cells purified by anchorage velocity-dependent separation method.METHODS：The lumbar nucleus pulposus from Wistar rats were digested by trypsin and type II collagenase.The first and second passages of cells were isolated and purified by anchorage velocity-dependent separation method during subculture.RESULTS AND CONCLUSION：The third passage of nucleus pulposus cells which were isolated and purified by anchorage velocity-dependent separation method appeared round or multi-angular with activity.Hematoxylin-eosin staining appeared homogeneous with blue nucleus and pink cytoplasm.The positive rate of type II collagen immunohistochemistry staining was 97% and rate of aggrecan immunohistochemistry staining was 95%.Transmission electron microscope results showed that there were many endoplasmic reaticulums,Golgi complexes,free ribosemes and few multilamellar bodies,but no chondriosome.The cell growth curve showed that after subculture,the third passage 3 of nucleus pulposus cells experienced 2 days of latent phase,3 days of increased logarithmic phase,then went to plateau phase.Anchorage velocity-dependent separation method is a practical and effective method for purifying the primary cultured rat nucleus pulposus cells.The third passage of cells harvested through this method is active and homogeneous.