细胞骨架中间纤维破坏对体外软骨细胞代谢功能的影响  

The effect of intermediate filament destruction in cytoskeleton on metabolic functions of chondrocytes in vitro

作　　者：王磊[1] 郭恒[1] 段王平[1] 卫小春[1] 

机构地区：[1]山西医科大学第二医院骨科,太原030001

出　　处：《中国药物与临床》2011年第7期745-747,共3页Chinese Remedies & Clinics

基　　金：国家自然科学基金面上项目(30872616);国家重点基础研究发展计划资助项目(2009CB526514)

摘　　要：目的探讨破坏中间纤维对体外软骨细胞代谢功能的影响。方法 2个月龄新西兰大白兔双膝关节全层软骨消化为原代软骨细胞,培养3d贴壁后,分为对照组和实验组,对照组用原代培养基继续培养,实验组在原代培养基中加入中间纤维破坏剂丙烯酰胺(终浓度为2.5mmol/L)。加药后第1、2天利用磷脂酰丝氨酸外翻分析法检测细胞早期凋亡率,第6天用细胞爬片苏木素-伊红(HE)染色观察细胞形态的改变,第3、6、9天用实时定量聚合酶链反应法测定软骨细胞Ⅱ型胶原、糖胺多糖以及基质金属蛋白酶(MMP)-13mRNA的表达量。结果实验组在加药后第2天软骨细胞的早期凋亡率高于对照组(P<0.05);和对照组相比较,实验组在加药后第6天HE染色观察到细胞形态不规则,呈多角形,胞核深染且分裂像增多,细胞基质减少;在加药后第3、6、9天检测到实验组Ⅱ型胶原和糖胺多糖的mRNA表达相对于对照组下降(P<0.05);在加药后第6、9天MMP-13mRNA表达显著增加(P<0.01)。结论中间纤维破坏后可以诱导软骨细胞的早期凋亡,致Ⅱ型胶原和糖胺多糖产生减少,而炎性因子MMP-13表达增多,可能是骨关节炎产生的一个重要环节。Objective To investigate the effect of intermediate filament destruction on metabolic functions of chondroeytes in vitro. Methods Five 2-month old New Zealand white rabbits were harvested for full-thickness cartilages of double knee joint under sterile conditions. The full-thickness cartilages were then digested into primary ehondrocytes in sequence by using 0.4% pronase and 0.025% type II collagen and cultivated for 3 days. After adhesion, chondrocytes were divided into control and experimental groups and successively cultured by using primary medium （90% DMEM/F12＋10% fetal calf serum）, with the experimental group additionally added intermediate filament destruc- tive agent-acrylamide （final concentration： 2.5 mmol/L）. The early apoptosis rate was detected by annexin V at 1 and 2 days after drug administration, change of cell morphology observed by HE staining of cell climbing sheet at 6 d, the ex- pression levels of type II collagen, glycosaminoglycans and MMP-13 mRNA determined by real time PCR at 3, 6 and 9 days. Results Compared with the control group, the experimental group had an early apoptosis rate of chondrocytes at 2 d after drug administration （P〈0.05）, irregular polygonal cell morphology, deep-stained nucleus, relatively increased division and decreased cell matrix with the HE staining at 6d; relatively decreased mRNA expressions of type II collagen and glycosaminoglycans at 3, 6 and 9 days （P〈0.05）. Significantly increased MMP-13 mRNA expression level at 6 d （P〈0.01）. Conclusion The early apoptosis of chondrocyte can be induced by intermediate filament destruction of vimentin, which may lead to decreased type II collagen and glycosaminoglycans and increased expression of inflammatory factor MMP-13. Therefore, the intermediate filament destruction of chondrocytes may be a major link of osteoarthritis generation.

关 键 词：软骨细胞 胶原Ⅱ型 基质金属蛋白酶类 细胞骨架 

分 类 号：R684.3[医药卫生—骨科学]