作　　者： (李大鹏）; (吴燕）; (黄永辉）; (蒋璐）; (孙继芾）; (岳佳伟）;

机构地区： 江苏大学附属医院,江苏镇江,212001

出　　处： 《山东医药》

摘　　要： 目的 建立一种体外分离、培养人髓核细胞(NPCs)的新方法,并探讨其意义.方法 采用0.2% Ⅱ型胶原酶消化法分离人正常NPCs,单层贴壁法进行传代培养,倒置显微镜下观察细胞形态.取第1、2、3代NPCs,采用CCK-8法检测细胞增殖能力(以吸光度值表示),连续检测12天.取第2代NPCs,采用免疫组化法检测Ⅱ型胶原(Col-2)、细胞角蛋白18(KRT-18)、KRT-19、低氧诱导因子1α(HIF-1α)、葡萄糖转运子1(GLUT-1)、Sox-9、聚集蛋白聚糖(ACAN)、CD24阳性表达率.结果 分离的NPCs呈多角形或短梭形,类似于软骨细胞;第4代以后,NPCs细胞突起延长,呈长梭形,生长缓慢,出现老化现象.NPCs生长曲线呈S形:1~2天细胞生长缓慢;3~7天细胞生长迅速,进入对数生长期;8天后进入平台期,细胞增殖缓慢.连续培养12天,第1、2、3代NPCs相同时间点吸光度值比较差异均无统计学意义(P均>0.05).第2代NPCs中Col-2、KRT-18、KRT-19、HIF-1α、GLUT-1、Sox-9、ACAN、CD24阳性表达率均≥85.6%(85.6%~91.2%).结论 单纯0.2% Ⅱ型胶原酶消化法及单层贴壁法可分离、培养人NPCs,并经免疫组化染色鉴定证实;传代3代以内的NPCs细胞形态及增殖能力均无明显改变,可作为椎间盘退变相关研究的种子细胞. 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.

分 类 号： [R681.5]