低强度脉冲超声促进人骨性关节炎软骨细胞合成细胞外基质

Low-intensity pulsed ultrasound promotes extracellular matrix synthesis of human osteoarthritis chondrocytes

目的探讨低强度脉冲超声(LIPUS)对人骨性关节炎(OA)软骨细胞的细胞外基质(ECM)的影响及相关作用机制。方法取膝关节置换术后废弃软骨组织,进行软骨细胞分离、培养、鉴定;取第2代软骨细胞随机分为OA对照组、30 m W/cm2LIPUS处理的OA组、30 m W/cm2LIPUS联合5μmol/L LY294002处理的OA组,除对照组外,其他组给予LIPUS刺激20 min/d×7 d;采用实时定量PCR法检测细胞中2型胶原蛋白(Col2)、聚集蛋白聚糖(aggrecan)、基质金属蛋白酶13(MMP-13)mRNA的水平,Western blot法检测细胞中Col2、aggrecan、MMP-13、Akt及磷酸化Akt(p-Akt)蛋白的表达。结果与OA对照组相比,LIPUS处理的OA组细胞内Col2、aggrecan mRNA和蛋白水平均增高,MMP-13表达降低,p-Akt蛋白的表达显著增加,Akt的表达则与OA对照组无显著性差异;与LIPUS处理的OA组细胞相比,LIPUS联合LY294002处理的OA组细胞中Col2、aggrecan mRNA和蛋白表达均降低,MMP-13表达增高,p-Akt蛋白的表达显著降低,Akt的表达则与LIPUS处理的OA组无显著性差异。结论 LIPUS促进人OA软骨细胞合成细胞外基质,并抑制其降解。

Objective To investigate the effect of low-intensity pulsed ultrasound（LIPUS） on the extracellular matrix synthesis of human osteoarthritis（OA） chondrocytes and explore the underlying mechanism.Methods Human osteoarthritis chondrocytes were collected from abandoned articular cartilage.Then the cells were cultured and identified by toluidine blue staining and immunocytochemical staining of type 2 collagen.The passage 2 cells were randomly divided into 3 groups：control OA group,30 m W/cm^2 LIPUS-treated OA group,30 m W/cm^2 LIPUS combined with 5 μmol/L LY294002-treated OA group.LIPUS treatment was performed for 20 minutes per day,totally 7 days.The mRNA levels of Col2,aggrecan and matrix metalloprotease 13（MMP-13） were determined by quantitative real-time PCR.The protein levels of Col2,aggrecan,Akt,p-Akt and MMP-13 were evaluated by Western blotting.Results Compared with the control OA group,the expressions of Col2 and aggrecan at both mRNA and protein levels significantly increased,and MMP-13 significantly reduced in the LIPUS-treated OA group.The p-Akt protein level was significantly elevated after LIPUS stimulation,but there was no significant difference in the Akt protein levels between the two groups.Moreover,LY294002,an inhibitor of PI3 K/Akt,significantly suppressed the biological effect activated by LIPUS.Conclusion LIPUS enhances the synthesis and inhibits the degradation of the extracellular matrix in human osteoarthritis chondrocytes.