右美托咪定对软骨细胞内ADAMTS5介导基质降解及相关因子表达的影响

Effects of dexmedetomidine on ADAMTS5 mediated matrix degradation in chondrocytes and expressions of related factors

目的 探讨右美托咪定(dexmedetomidine, DEX)干预对体外培养软骨细胞和相关因子表达的影响及可能分子机制。方法 体外培养C28/I2正常人软骨细胞,选取1μmol/L的DEX分别干预24、48 h。观察软骨细胞形态及细胞密度;用免疫荧光技术检测各组软骨细胞内人金属肽酶含血小板反应蛋白基元5(a disintegrin and metalloproteinase with thrombospondin motifs 5,ADAMTS5)蛋白表达;用RT-PCR技术检测各组软骨细胞内软骨基质相关因子Adamts5、蛋白多糖(Acan)、多功能蛋白聚糖(Vcan)、弗林蛋白酶(Furin)、前蛋白转化酶枯草溶菌素6(Pcsk6)、Ⅰ型胶原α1链(Col1a1)、Ⅱ型胶原α1链(Col2a1)、Ⅹ型胶原α1链(Col10a1)和Y染色体性别决定基因相关高迁移率族蛋白盒9(Sox9)等的mRNA表达。应用慢病毒转染技术构建Adamts5基因敲除软骨细胞,并分别对Adamts5基因敲除软骨细胞给予DEX处理24、48 h,并用RT-PCR技术检测各组细胞内Acan、Vcan、Col1a1、Col2a1、Col10a1等基因mRNA的表达。结果 经1μmol/L DEX干预24、48 h后,软骨细胞形态和大小未发生明显变化,但细胞密度略有增加;免疫荧光染色显示,ADAMTS5蛋白表达呈先增高后降低的趋势;RT-PCR结果显示,培养24、48 h,Adamts5基因mRNA表达差异具有统计学意义(P=0.032),随干预时间延长,表达呈先增高后降低;Pcsk6基因的mRNA表达变化趋势与Adamts5相同,而Acan的表达趋势与Adamts5相反。敲除Adamts5的软骨细胞中,Adamts5 mRNA显著降低而Vcan mRNA表达增加;随后用DEX对其干预24、48 h,Acan和Vcan的表达均逐渐降低,但与正常软骨细胞相比差异无统计学意义。结论 DEX可能通过Pcsk6激活ADAMTS5酶原,从而促进软骨细胞内的蛋白聚糖降解。

Objective To investigate the effects of dexmedetomidine(DEX) intervention on the expressions of chondrocytes and related factors in vitro and its possible molecular mechanisms. Methods C28/I2 normal human chondrocyte lines were cultured in vitro, and dexmedetomidine at the concentration of 1 μmol/L was selected to intervene for 24 h and 48 h, respectively. The morphology and cell density of chondrocytes were observed after DEX culture at different time points. Immunofluorescence technique was used to detect the expression levels of disintegrin and metalloproteinase with thrombospondin motifs 5(ADAMTS5) in chondrocytes in each group. The expression levels of Adamts5, aggrecan(Acan), versican(Vcan), Furin, proprotein convertase subtilisin/kexin type 6(Pcsk6), collagen type Ⅰ alpha 1(Col1a1), collagen type Ⅱ alpha 1(Col2a1), collagen type X alpha 1(Col10a1), and SRY2 related high mobility group box gene9(Sox9) were detected by RT-PCR. Adamts5 gene knockout chondrocytes were constructed by lentivirus transfection technology and treated with DEX;RT-PCR was used to detect the effects of DEX on the expression levels of Acan, Vcan, Furin, Pcsk6 and Sox9 after Adamts5 gene knockout. Results After 24 and 48 h of intervention with 1 μmol/L DEX, the morphology a nd size of chondrocytes did not change significantly, but the cell density increased slightly. Immunofluorescence assay showed that the expression of ADAMTS5 increased at first and then decreased after DEX treatment for 24 and 48h, respectively(P=0.032). RT-PCR results showed that with the extension of intervention time, the expression of Adamts5 first increased and then decreased. The expression difference between 48 and 24 h after culture was statistically significant(P=0.032). The change trend of Pcsk6 was the same as that of Adamts5, while the change trend of Acan expression was opposite that of Adamts5. Chondrocytes knocked out Adamts5 gene and intervened with DEX for 24 and 48 h. The results of RT-PCR showed that the expression of Pcsk6 decreased while that of Acan increased and the changes were significant. Conclusion Dexmedetomidine may activate ADAMTS5 zymo gen through Pcsk6, thereby promoting proteoglycan degradation in chondrocytes.