胶原酶优化消化法对退变椎间盘纤维环细胞分离培养的作用

Effection of optimized digestion of collagenase on the culture of annulus fibrosus cells of degenerated intervetebral disc

目的通过观察不同浓度Ⅰ、Ⅱ型胶原酶对退变椎间盘纤维环组织消化效果,探讨适合于纤维环细胞分离培养的优化消化法。方法收集16例退变椎间盘纤维环组织,Ⅰ、Ⅱ型胶原酶单独消化并在0.1%,0.05%,0.01%浓度下联合消化。消化4、8、16、24 h时倒置显微镜下计算纤维环细胞数,台盼蓝染色检查其活力,并观察其生长情况。结果Ⅰ、Ⅱ型胶原酶联合消化细胞数多于单独消化细胞数。联合消化4、8 h时,细胞数随胶原酶消化浓度增加而增多,16、24 h时极低浓度组纤维环细胞数逐渐增多,细胞活力维持在较高水平。结论极低浓度Ⅰ、Ⅱ型胶原酶优化消化法能节省酶量,对细胞损伤小,更适合纤维环细胞分离培养。

Objective To explore the optimized digestive method of collagenase to annulus fibro-sus（AF） cells by observing the digestive effects of type I,II collagenase in different concentrations on AF of degenerated intervetebral disc. Methods 16 AF samples from human herniated intervertebral disc were collected and digested by type I,II collagenase individually or combined in different con-centration. The AF cells were counted with inverted microscope and their activities were checked with trypan blue staining at 4 h、8 h、16 h、24 h after digestion. The growth of AF cells was also observed. Results The amount of AF cells with combined collagenases was more than that seperat-edly in the identical concentration. With combined collagenases in 4, 8 hours, the higher concentra-tion of collagenases,the more AF cells. The amount of cells in extremely low concentration of colla-genases increased at 16 h、24 h,and their activities kept in higher level. Conclusion The optimized digestion at extremely low concentration of typeⅠand Ⅱcollagenase combination could save enzyme, and is less harmful to AF cells,which is more adapted to separate and culture of AF cells.