山东医药2017，Vol.57Issue(32)：1-4,前插1,5.DOI:10.3969/j.issn.1002-266X.2017.32.001
人髓核细胞分离、传代培养方法的建立及意义
Establishment and significance of separation and culture of human nucleus pulposus cells
摘要
Abstract
Objective To establish a new method for isolation and culture of human nucleus pulposus cells (NPCs) in vitro and to explore its significance.Methods Human normal NPCs were isolated by type Ⅱ collagenase digestion, then were cultured and expanded in monolayer.Cell morphology was observed by inverted microscope;the proliferation ability (expressed as absorbance value) of passage 1, passage 2 and passage 3 NPCs were detected by CCK-8 assay for consecutive 12 days;the positive expression rates of type Ⅱ collagen (Col-2), cytokeratin 18 (KRT-18), KRT-19, hypoxia-inducible factor-1α(HIF-1α), glucose transporter 1 (GLUT-1), Sox-9, aggrecan (ACAN), and CD24 in passage 2 NPCs were detected by immunohistochemistry.Results The isolate human NPCs were polygonal or short spindle, similar to chondrocytes.However, the nucleus pulposus cells after passage 4 grew slowly, the protrusion of cells extended, and the cells were long spindle shape, all of these indicated the cells were aging.The growth curve of NPCs was S-shaped, cells grew slowly in 1-2 days, then grew into the logarithmic growth phase in 3-7 days, and after 8 days the cells proliferated slowly and entered the plateau.After consecutive 12-day culture, the difference of absorbance values at 450 nm among passage 1, passage 2 and passage 3 NPCs was not statistically significant at the same time point (all P>0.05).Immunohistochemical staining showed: the positive rates of Col-2, KRT-18, KRT-19, GLUT-1, HIF-1α, Sox-9, ACAN, and CD24 in passage 2 NPCs were no less than 85.6% (85.6%-91.2%).Conclusions Human NPCs can be effectively isolated by 0.2% type Ⅱ collagenase digestion method, and can be cultured by monolayer adherence method.The cells we cultured were identified as nucleus pulposus cells by immunohistochemistry.There are no significant changes in the morphological and proliferation abilities of NPCs within 3 generations, and could be used as seed cells for study of intervertebral disc degeneration.关键词
髓核细胞/Ⅱ型胶原酶消化法/单层贴壁法/细胞培养/椎间盘退变Key words
nucleus pulposus cells/type Ⅱ collagenase digestion/monolayer adherence/cell culture/intervertebral disc degeneration分类
医药卫生引用本文复制引用
李大鹏,吴燕,黄永辉,蒋璐,孙继芾,岳佳伟..人髓核细胞分离、传代培养方法的建立及意义[J].山东医药,2017,57(32):1-4,前插1,5.基金项目
国家自然科学基金资助项目(81601931) （81601931）
江苏省自然科学基金资助项目(BK20150475). （BK20150475）
