毫米波干预软骨细胞β-catenin活性作用机制研究

The Role of Millimeter-wave Intervention for Chondrocyte in β-catenin Activity Mechanism

为了探讨毫米波干预软骨细胞β-catenin活性的作用机制,采集雄性4周龄SD大鼠膝关节软骨,采用机械-II型胶原酶消化,建立体外培养软骨细胞,甲苯胺蓝染色鉴定。第2代软骨细胞培养48h,用不含血清DMEM饥饿24h,更换为10%FBSDMEM,随机分为4组:正常组、实验1组(毫米波干预30min)、实验2组(毫米波干预60min)和实验3组(毫米波干预120min)。各实验组毫米波干预后,培养24h,II型胶原免疫组化观察各组软骨细胞的功能,RT-PCR检测β-catenin、GSK-3β、CKIεmRNA表达,WesternBlot检测β-catenin、GSK-3β、CKIε蛋白表达水平。结果表明,机械-II型胶原酶消化法,能够成功建立体外培养的软骨细胞,第2代软骨细胞培养3d,甲苯胺蓝染色可见软骨细胞内呈紫红色异染颗粒,细胞周围也出现紫红色异染颗粒,细胞核呈深蓝色,以圆形、椭圆形为主;干预前后,各组软骨细胞II型胶原免疫组化均可见胞浆呈棕黄色,核呈圆形不着色;实验2组、实验3组软骨细胞β-catenin、CKIεmRNA表达显著高于正常组(P<0.01),实验2组、实验3组软骨细胞β-catenin、CKIεmRNA表达显著高于实验1组(P<0.01),实验2组、实验3组软骨细胞β-catenin、CKIε蛋白表达水平显著高于正常组(P<0.01),实验2组、实验3组软骨细胞β-catenin、CKIε蛋白表达水平显著高于实验1组(P<0.01);实验2组、实验3组软骨细胞GSK-3βmRNA表达显著低于正常组(P<0.01),实验2组、实验3组软骨细胞GSK-3βmRNA表达显著低于实验1组(P<0.05),实验2组、实验3组软骨细胞GSK-3β蛋白表达水平显著低于正常组(P<0.01),实验2组、实验3组软骨细胞GSK-3β蛋白表达水平显著低于实验1组(P<0.01)。毫米波的能量可通过生物体的谐振而被吸收,生物系统谐振吸收电磁能量后又产生了不属于温度变化的生物学效应,干扰生物体的信号传导,动态调节软骨细胞的生物学功能,毫米波作用机制可能是通过诱导软骨细胞转录合成β-catenin、CKIε,抑制GSK-3β表达,从而促进β-catenin合成、抑制分解,提高软骨细胞内β-catenin的活性,并且能维持软骨细胞表型的稳定。

To investigate the effects of millimeter wave treatment on the activation of β-catenin in chondrocytes, knee articular cartilages of four-week-old SD rats were extracted and digested with mechanical type II collagens. Cells were identified with toluidine blue staining and the secondary generation chondrocytes were cultured for 48 h, and kept hunger for 24 h in serum-free Dulbecco＇s Modified Eagle Media （DMEM）, which was then replaced by 10% FBS DMEM. The cells were randomly divided into control group, experimental 1 group （receiving millimeter wave signal for 30 minutes）, experimental 2 group （receiving millimeter wave signal for 60 minutes） and experimental 3 group （receiving millimeter wave signal for 120 minutes）. Treated with millimeter wave, the secondary generation chondrocytes were cultured for 24 h, and the cell function was detected by type II collagen immunohistochemistry, the levels of β-catenin, GSK-3,8 and CKIε were detected using RT- PCR and western blotting. With mechanical and chemical isolation methods, the well-distributed second generation cells were shown with purplish red metachromatic granules in cytoplasm, blue nucleus and surrounding of metachromatic granules, which were primarily round and oval. Before and after the intervention of the millimeter wave, for type II collagen immunohistochemistry of all groups, nucleus appeared colorless with brown-yellow cytoplasm, most of which were round. After intervention, mRNA expression of β- catenin and CKIε in cells of experimental 2 and 3 groups were significantly higher than the control group （P〈0.01）, mRNA expression of β-catenin and CKIε in cells of experimental 2 and 3 groups were significantly higher than experimental 1 group （P〈0.01）, the protein levels of phosphorylated β-catenin and CKIc in cells of experimental 2 and 3 groups were significantly higher than the control group （P〈0.01）, the protein levels of phosphorylated β- catenin and CKIε in cells of experimental 2 and 3 groups were significantly higher than experimental 1 group （P〈0.01）. mRNA expression of GSK-3β in cells of experimental 2 and 3 groups were significantly lower than the control group （P〈0.0 1）, mRNA expression of GSK-3β in cells of experimental 2 and 3 groups were lower than experimental 1 group （P〈0.05）, the protein levels of phosphorylated GSK-3β in cells of experimental 2 and 3 groups were significantly lower than the control group （P〈0.01）, the protein levels of phosphorylated GSK-3β in cells of experimental 2 and 3 groups were significantly lower than experimental 1 group （P〈0.01）. The energy of the millimeter wave can be absorbed by the organism through resonance. In turn, the energy can be transferred to interfere with the signal transduction in the organism, dynamically regulate metabolism, and regulate biological functions of chondrocytes. Millimeter wave intervention β-catenin activity in chondrocytes may be improved through effectively regulating the expression and activation of β-catenin, CKIε and inhibiting the expression and activation of GSK-3β, promoting synthesis of β-catenin, inhibiting decomposition, and at the same time, the stable features of chondrocytes are maintained.