周期性张应变力对幼年和成年及骨关节炎软骨细胞产生糖胺多糖的影响

Effects of cyclic tensile strain on expression of GAG in rabbit chondrocytes of juvenile,adult and OA groups

目的观察周期性张应变力(cyclic tensile strain,CTS)对体外培养的幼年、成年及骨性关节炎(Osteoarthritis,OA)兔软骨细胞产生糖胺多糖(Glycosaminoglycans,GAG)的影响。方法取2月龄雄性新西兰大白兔3只作为幼年组,5月龄雄性新西兰大白兔10只,随机分2组(即成年组与OA组),每组5只。OA组采用前交叉韧带切断术(ACLT)制作OA动物模型,成年组仅行双膝关节切开,但不行ACLT。成年组及OA组动物于术后20周空气栓塞处死,取左侧膝关节标本分别做大体评分及Mankin`s评分观察OA造模情况。将三组兔膝软骨细胞进行消化分离及体外培养。将每个组原代软骨细胞均分别培养于2个BioFlex6孔培养板上,随机分为对照组细胞和加载组细胞,每组6个样本,同置于培养箱内,加载组每天CTS(sin10%,0.5Hz,6h/次)作用6h,对照组不予特殊处理。加载组与对照组在首次加载后24h、48h与72h分别吸取培养细胞上清液,阿尔新蓝染色沉淀法测定上清液GAG含量,比较各组GAG分泌的变化。结果①实验组动物关节软骨明显退变,大体评分及Mankin's评分均明显高于对照组(P<0.05);②与对照各组细胞相比,幼年、成年及OA组细胞进行加载后其GAG的分泌水平随时间均升高(P<0.05);且各细胞在CTS干预前后GAG分泌随时间变化趋势存在统计学差异(P<0.05)。结论 Flexercell-4000力学加载系统可对体外培养的软骨细胞施加精确的力学负荷加载,并从刺激各类软骨细胞产生蛋白多糖。与正常细胞相比,OA软骨细胞对力学刺激的响应特点发生了较明显的变化。

Objective To observe the different effects of cyclic tensile strain on expression of GAG of juvenile,adult and OA groups of rabbit chondrocytes in vitro. Methods Choose 3 New Zealand white rabbits of two months as juvenile group,and 10 New Zealand white rabbits of three months were divided into two groups randomly as adult and OA group,each group 5. The animal model of OA was induced by anterior cruciate ligament transection in animals of OA group ,while similar process of operation（ without ACLT） performed on ones of adult group. Both groups（ adult and OA group） were evaluated 20 weeks larer. The juvenile,adult and OA chondrocytes were cultivated in vitro,each group were randomly divided into 2 groups,control group and CTS group. And then,every CTS group was stimulated with CTS（ 0. 5Hz,sin10% ,6 h/d,3 days） by the Flexercell4000 Tension System. At the time of 24 h、48 h、72 h,we collected the medium of the cultured chondrocytes stained with Alcian blue precipitation method to analyse the expression of GAG of the chondrocytes. Results ①The knee joints of OA group had obvious degeneration of articular cartilage,and the Mankins grades of macroscopic was significantly higher than adult group（ P 0. 05） . ②CTS （ sin10% ,0. 5Hz,6 h/d） can stimulate chondrocytes of different groups,including juvenile group,adult group and OA group,secretion more GAG within 72 h,while the trends of GAG secretion over time showed significant differences between them. Conclusion Flexercell-4000 Mechanical loading system can stimulate many kinds of chondrocytes secretion more GAG by loading mechanical stimulation. Compared to adult and juvenile cells,OA cells appeared sigficant difference in characteristic of mechanical response.