Ⅰ型神经纤维瘤病萎缩区椎体终板软骨细胞体外分离培养及表型鉴定  

Culture and identification of chondrocytes isolated from the vertebral endplate of patients with type Ⅰ neurofibromatosis associated with atrophic changes in vitro

作　　者：刘学光[1,2] 邱 勇[3] 孙振中[1,2] 钱邦平[3] 王守丰[3] 

机构地区：[1]无锡市第九人民医院骨科 [2]无锡市手外科医院,江苏省无锡市214064 [3]南京大学附属南京鼓楼医院脊柱外科,江苏省南京市210008

出　　处：《中国组织工程研究》2014年第46期7396-7400,共5页Chinese Journal of Tissue Engineering Research

基　　金：江苏省自然科学基金(BK 2010109)~~

摘　　要：背景:以往主要是针对肋软骨、关节软骨、鼻中隔软骨、耳软骨等透明软骨进行研究,而对人椎体终板软骨的体外培养尚不多见。目的:探讨Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸患者萎缩区椎体终板软骨细胞体外分离培养的可行性,并对其生物学特性进行观察。方法:对术中取得的7例Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸患者顶椎区椎体终板软骨采用两步酶消化结合组织块法进行体外分离培养。细胞传代后倒置相差显微镜下观察细胞形态;Ⅱ型胶原免疫组化染色对软骨细胞进行鉴定;MTT比色法测定细胞增殖情况,描绘第2代软骨细胞的生长曲线。结果与结论:软骨组织培养2周后周围开始有软骨细胞爬出,再经1周左右可以传代。随着传代次数的增加,软骨细胞逐渐由多角形、圆形、三角形和不规则形变为梭形,折光性下降。Ⅱ型胶原免疫组化染色可见细胞核周围被染成棕褐色。MTT比色法检测显示,第2代终板软骨细胞的生长曲线近似倒"S"形,在第4-7天进入快速增殖期,细胞呈对数生长;到第8-13天达平台期,细胞增殖停止;从第14天开始出现生长抑制。结果显示采用两步酶消化结合组织块法能够培养出纯度高,活性好的终板软骨细胞。第2代终板软骨细胞在第4-7天活力最好,增殖力旺盛,可以作为研究Ⅰ型神经纤维瘤病伴萎缩性脊柱侧凸发病机制的观察点。BACKGROUND：Previous studies have mainly focused on costal cartilage, articular cartilage, nasal septal cartilage, and auricular cartilage, but in vitro culture of human vertebral endplate cartilage is stil rarely reported. OBJECTIVE： To discuss the feasibility of culture of vertebral endplate chondrocytes from type I neurofibromatosis associated with scoliosis patientsin vitro and to study the biological characters of the chondrocytes. METHODS： Through two-step enzymatic digestion and tissue culture, the chondrocytes from the vertebral endplate of seven type I neurofibromatosis patients isolated and cultured in monolayer and passaged to observe the changes of cel morphology under inverted phase contrast microscope. Colagen type II expression was detected by immunocytochemistry to identify whether the cels had chondrocyte characters. The growth kinetics was detected by using MTT colorimetric assay to draw the growth curve of passage 2 chondrocytes. RESULTS AND CONCLUSION：A few chondrocytes crawled from the cartilage after 2 weeks culture and cels were passaged at 3 weeks. Along with passage going on, the phenotype of chondrocytes was changed from＆amp;nbsp;polygonal, round, triangle, and irregular shapes to fusiform. The colagen type II expression in passage 2 cels was positive by immunohistochemical staining. MTT test showed the growth curve of the passage 2 chondrocytes presented a transverse “S”. Cels were found logarithmic growth at days 4-7, reached platform stage at days 8-13, and decreased at day 14. It is an effective and simple procedure by two-step enzymatic digestion and tissue explant method to culture vertebral endplate chondrocytes with high purity and good viability from type I neurofibromatosis patients associated with scoliosisin vitro. Passage 2 chondrocytes from the vertebral endplate exhibit the best viability at days 4-7, which can be used as targets for research of pathogenesis of type I neurofibromatosis with atrophic scoliosis.

关 键 词：组织构建 软骨细胞 神经纤维瘤病 细胞培养 终板 表型 江苏省自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]