微管蛋白破坏对软骨细胞代谢功能的影响

Disassembly of tubulin cytoskeleton disrupts the homeostasis of articular cartilage chondrocytes

目的 探讨微管蛋白破坏对体外关节软骨细胞代谢功能的影响.方法 2月龄新西兰白兔8只,处死后取双膝关节全层软骨,采用常规0.4%链酶菌蛋白酶和0.025%Ⅱ型胶原酶依次消化为软骨细胞培养3 d贴壁后,分为对照组和实验组,对照组继续用原代培养基（90%DMEM/F12±10%胎牛血清）培养,实验组在原代培养基中加入微管蛋白破坏剂秋水仙素（终浓度为0.1μmol/L）.加药后第1、2天用Annexin-Ⅴ/PI流式细胞术检测软骨细胞早期凋亡率,加药后第6天细胞爬片HE染色观察细胞形态的改变,加药后第3、6、9天取细胞用实时定量荧光反转录聚合酶链式反应法测定软骨细胞Ⅱ型胶原、蛋白多糖以及MMP-13 mRNA的表达量,同时在加药后第3、6、9天取细胞上清液,用ELISA法和阿尔新蓝法检测各组上清液中Ⅱ型胶原和蛋白多糖的含量.结果 实验组第2天软骨细胞早期凋亡率明显高于对照组（P＜0.05）;与对照组比较,实验组第6天软骨细胞呈不规则多角形,胞核深染且分裂象增多,细胞基质减少,实验组第3、6、9天软骨细胞Ⅱ型胶原和蛋白多糖mRNA表达量较对照组均明显降低（P＜0.05）,第6、9天实验组软骨细胞MMP-13mRNA表达较对照组明显增高（P＜0.01）,同时实验组第3、6、9天上清液中的Ⅱ型胶原和蛋白多糖含量均明显低于对照组（P＜0.05或P＜0.01）.结论 微管蛋白破坏可致软骨细胞早期凋亡,导致体外培养软骨细胞Ⅱ型胶原、蛋白多糖等基质成分合成减少,炎性因子MMP-13的表达增多.

Objective To investigate the influence of tubulin disassembly on the in vitro metabolism of articular chondrocytes. Methods Eight New Zealand rabbits aged 2 months were sacrificed by air embolism. The full-thickness cartilages were harvested from both knees under sterile conditions. Then 0. 4% pronase and 0. 025% Ⅱ collagenase were used to digest for primary chondroeytes. The cells were cultured for 3 days after attachment and then divided into the control and experimental groups. The control group continued culturing with primary medium and the experimental group with colchieine, a tubulin destructive agent, at a final concentration of 0. 1 ~mol/L. At Days 1 ＆ 2, the early apoptosis of two cell groups was assayed with phosphatidylserine （Anuexin V）. At Day 6, the morphological changes of cells were observed by hematoxylin and eosin staining. At Days 3,6 ＆ 9, the expression levels of type 1I collagen, proteoglycan and MMP （matrix metalloproteinase）-13 mRNA were measured by real time quantitative fluorescent reverse transcription-polymerase chain reaction （real time RT-PCR）. Meanwhile at Days 3, 6 ＆ 9, the cultured supernatant of each group was obtained to detect the contents of type ]1 collagen and proteoglycan by the methods of ELISA （ enzyme-linked immunosorbent assay） and Alcian blue. Results At Day 2, the early cell apoptosis rate of the experimental group was higher than that of the control group （ P 〈 0. 05 ）. At Day 6 post-dosing, the experimental cell morphology was found to be irregular and polygonal. Hyperchromatic nucleus and division phase increased while matrix cell decreased. At Days 3, 6 ＆ 9, the expressions of collagen and proteoglycan mRNA in the experimental group decreased versus those in the control group （ P 〈 0. 05）. The level of MMP-13 mRNA showed no significant changes in the control group at Day 3. And it significantly increased at Days 6 ＆ 9 （P 〈 0. 01 ）. Meanwhile, type Ⅱ collagen and glycosaminoglycan were significantly lower in the experimental group at Days 3, 6 ＆ 9 than those in the control group （ P 〈 0. 05 ）. Conclusion A disassembly of tubulin can induce early apoptosis of chondrocytes. The synthesis and secretion of type Ⅱ collagen and proteoglycan decrease while there is an elevated expression of MMP-13. The degeneration of cartilage cells and their disrupted synthesis and catabolism result in a serious decline in biological functions.