人骨关节炎软骨细胞的体外分离与培养  

In vitro isolation and culture of chondrocytes from human osteoarthritis patients

作　　者：魏钰[1] 魏民[1] Wei Yu;Wei Min(Chinese PLA General Hospital, Beijing 100000, China)

机构地区：[1]中国人民解放军总医院

出　　处：《中国组织工程研究》2019年第25期4056-4061,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81473710,81873175),项目负责人:魏民~~

摘　　要：背景:大量针对骨关节炎细胞水平的研究仍集中在动物模型关节软骨细胞的体外培养。动物模型引发的退变效应与人骨关节炎的发生并不完全一致。如何利用人骨关节炎软骨组织构建体外细胞模型并研究其特征性蛋白变化趋势,是模拟体内骨关节炎软骨退变过程的关键。目的:探讨人骨关节炎软骨细胞的体外分离、培养方法,观察原代至第3代人骨关节软骨细胞的形态学特性和相关蛋白表达变化。方法:取6例因重度骨关节炎行关节置换术患者的废弃软骨组织(获得中国人民解放军总医院医学伦理委员会批准,伦理批号为S2017-23-7),男2例,女4例;年龄62-72岁,平均(68.3±3.39)岁,采用一步酶消化法(Ⅱ型胶原酶)分离培养人骨关节炎软骨细胞,并进行传代培养,从而构建体外人骨关节炎软骨细胞培养体系。倒置相差显微镜观察各代细胞形态;苏木精-伊红染色、甲苯胺蓝染色及Ⅱ型胶原免疫荧光染色方法进行细胞鉴定。采用Western blot法检测各代软骨细胞于70%融合率时Col2a、Aggrecan与MMP-13的表达。结果与结论:经过Ⅱ型胶原酶消化后,培养1周左右可见组织块周围爬出散在的细胞,3周左右可传代进行下一步研究;形态学、苏木精-伊红染色、甲苯胺蓝染色与Ⅱ型胶原免疫荧光染色证明培养出的细胞为人软骨细胞。第3代软骨细胞Col2a、Aggrecan的相对表达量与原代比较有明显降低(P<0.01),且随着培养代数增加表达逐渐降低;MMP-13随着培养代数的增加表达逐渐增强(P <0.01)。结果表明,Ⅱ型胶原酶一步消化法可成功从标本中分离出骨关节炎软骨细胞并传代培养。体外培养的骨关节炎软骨细胞去分化特性随着传代次数增加而逐渐增强,功能性蛋白表达整体下降。3代以内的软骨细胞符合人骨关节炎时软骨退变的表现,可能是用于实验研究的最佳选择。BACKGROUND: Numerous studies addressing cells in osteoarthritis still focus on the in vitro culture of articular chondrocytes in animal models. Degeneration in the animal models is not completely consistent with that in human osteoarthritis. How to construct an in vitro cell model using chondrocytes from human osteoarthritis and to study changing trend of its characteristic proteins is the key to simulating the cartilage degeneration in human osteoarthritis in vivo.OBJECTIVE: To investigate the in vitro isolation and culture of human chondrocytes from osteoarthritis patients, to observe the morphological characteristics of human osteoarthritis chondrocytes from primary to third generation, and to study the changes in biological characteristics of chondrocyte-related proteins in different generation.METHODS: The study protocol was in line with the ethic requirements of Chinese PLA General Hospital with the approval No. S2017-23-7.Six cases undergoing arthroplasty for severe osteoarthritis(2 males and 4 females, age 62-72 years old with a mean age of(68.3±3.39) years)were enrolled. The chondrocytes from abandoned cartilage tissue in these patients were isolated and cultured by one-step enzymatic digestion(type Ⅱ collagenase), and then subcultured, so as to construct an in vitro chondrocyte culture system for osteoarthritis. The morphology of cells at different generations was observed by inverted phase contrast microscope. Hematoxylin-eosin staining, toluidine blue staining and type Ⅱ collagen immunofluorescence staining were used for cell identification. Western blot was used to detect the expressions of Col2 a, Aggrecan and matrix metalloproteinase-13 in each generation of chondrocytes at the fusion rate of 70%.RESULTS AND CONCLUSION: After digestion using type Ⅱ collagenase, the cells scattered around the tissue mass could be observed at about1 week of culture, and these cells could be subcultured for further study after about 3 weeks. Morphological observation, hematoxylin-eosin staining, toluidine blue

关 键 词：骨关节炎 关节置换 人软骨细胞 一步酶消化法 Ⅱ型胶原酶 Ⅱ型胶原 聚集蛋白聚糖 国家自然科学基金 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]