中间纤维对兔关节软骨细胞基质合成代谢的影响

Effects of intermediate filament cytoskeleton on the matrix anabolism of rabbit articular chondrocytes in vitro

目的观察细胞骨架中间纤维破坏对培养的兔关节软骨细胞基质合成代谢的影响。方法1个月龄新西兰白兔，无菌条件下切取双膝关节软骨，酶解法分离软骨细胞，接种于6孔板培养待细胞贴壁后弃去培养液并随机分为2组。对照组用DMEM／F12正常培养，中间纤维破坏组（简称IF破坏组）利用含5mmol／L丙烯酰胺的DMEM／F12培养，使细胞骨架中间纤维破坏。分别在换液后第4天和第7天以阿尔新蓝法检测2组细胞上清液中糖胺多糖（GAG）含量，同时检测上清液中羟脯氨酸含量以评估2组细胞胶原的合成量。结果两组软骨细胞在换液后第4天和第7天时上清液中GAG含量差异无统计学意义（P〉0．05）；但2组上清液中GAG含量均随培养时间的延长而升高（P〈0．05）。IF破坏组上清液中羟脯氨酸含量在换液后第4天时与对照组差异无统计学意义（P〉0．05），而第7天时明显低于对照组（P〈0．05）；对照组第7天上清液中羟脯氨酸含量较第4天有增高趋势，但统计学检验差异无统计学意义（P〉0．05），而IF破坏组第7天上清液中羟脯氨酸含量与第4天相同。结论软骨细胞GAG的合成具有时间累积效应，破坏中间纤维对软骨细胞GAG的合成没有影响，而破坏中间纤维可降低离体培养的关节软骨细胞羟脯氨酸的合成，表明中间纤维在软骨细胞胶原的合成中有着重要作用。

Objective To investigate the effects of disruption of intermediate filament cytoskeleton on the matrix anabolism of rabbit articular chondrocytes in vitro. Methods One-month-old New Zeal- and rabbits were killed and their knee joints were taken out under the aseptic condition. The chondrocytes were isolated by enzymolysis method and inoculated into a 6-well plate. The medium was abandoned after cell attachment and the cells were divided into two groups randomly. The cells in control group were cultured with DMEM/F12,while those in intermediate filament disruption group （IF disruption group） cultured in the media containing 5 mmol/L acrylamide. The glycosaminoglycan （GAG） content was determined by Alician Blue method, and the hydroxyproline content was measured to estimate collagen synthesis in the supernatant when medium was changed after 4 and 7 days. Results There was no significant difference in GAG content in the supernatant between the two groups with medium change after 4 and 7 days （P 〉 0.05）. The GAG content in the two groups were all significantly increased with the culture time （P 〈 0. 05 ）. The hydroxyproline content in IF disruption group showed no significant change at the 4th day after medium replacement （P 〉 0.05 ）, but significantly decreased at the 7th day as compared with the control group （P 〈 0.05 ）. The hydroxyproline content in control group had an increased tendency at the 7th day after medium replacement, but showed no significant difference compared that at the 4th day （ P 〉 0.05 ）. There was no significant change in the hydroxyproline content in the supernatant of IF disruption group between the 7th day and 4th day. Conclusion The GAG synthesis of chondrocytes has time cumulative effect,however,disruption of intermediate filament has no effect on the GAG synthesis. The disruption of the intermediate filament can decrease the collagen synthesis of chondrocytes in vitro, suggesting that the intermediate filament is necessary for the collagen synthesis of articular chondrocytes.