Effect of osteopontin on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes

doi:10.3969/j.issn.2095-4344.2016.42.002

Abstract

Abstract:

BACKGROUND: Both osteopontin and hyaluronic acid involve in the pathological process of osteoarthritis, resulting in the abnormal expression levels of various cytokines and enzymes. However, the relationship between the high expression of osteopontin and hyaluronic acid in chondrocytes remains unclear.
OBJECTIVE: To investigate the effect of osteopontin on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes in vitro by modulating the level of osteopontin.
METHODS: Chondrocytes from human knee osteoarthritic cartilage were cultured in vitro, and were then divided into three groups: blank control group without any treatment; osteopontin group and and pontin siRNA group were treated with 1 mg/L recombinant human osteopontin and osteopontin siRNA, respectively. Expression levels of osteopontin, hyaluronic acid synthase 1, 2 and 3 mRNA were detected by real-time PCR, and the levels of hyaluronic acid were measured using ELISA.
RESULTS AND CONCLUSION: Compared with the blank control group, the mRNA expressions of hyaluronic acid synthase 1, 2 and 3 were remarkably increased in the osteopontin group, while siRNA made the significantly inhibitory effects on the hyaluronic acid synthase 1, 2 and 3 mRNA expressions (P < 0.05). The level of hyaluronic acid in chondrocytes in the osteopontin group was significantly higher than that in the other two groups (P < 0.05). Our results suggest that osteopontin induces the synthesis of hyaluronic acid in osteoarthritic chondrocytes through upregulating the hyaluronic acid synthases expression levels.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteopontin, Chondrocytes, Hyaluronic Acid, Osteoarthritis, Tissue Engineering

CLC Number:

R318

Luo Wei1, Zhang Fang-jie1, Li Yu-sheng1, Lei Guang-hua1, 2. Effect of osteopontin on the expression of hyaluronic acid in human knee osteoarthritic chondrocytes[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(42): 6244-6251.

Figures/Tables 1

2.1 人膝骨性关节炎软骨细胞形态及骨桥蛋白siRNA转染鉴定 ①原代骨关节炎软骨细胞贴壁后，成团生长，以此为中心向四周扩散生长，软骨细胞多呈椭圆形、短梭形或三角形(图1A)；②抗Ⅱ型胶原抗体1∶1 000染色(图1B)；③荧光显微镜示大多数软骨细胞已转染siRNA(>90%)(图1C)；④MTT检测示各组间细胞生存率差异无显著性意义(P > 0.05)(图1D)；⑤mRNA鉴定示mRNA完整(图1E)；⑥Real timePCR结果显示：与NC-siRNA组对比，各组siRNA均显著降低骨桥蛋白mRNA表达(P < 0.05)，以siRNA-1最为明显(图1F)，选取siRNA-1作为后续试验干预序列。 2.2 标准曲线绘制及分析 Real time PCR 熔解曲线提示，内参基因β-Actin及目的基因骨桥蛋白、透明质酸合成酶1、透明质酸合成酶2、透明质酸合成酶3的熔解曲线呈单峰且熔解温度均一(图2A-E)，扩增曲线均接近S型曲线。 2.3 各组间骨桥蛋白mRNA、骨桥蛋白蛋白、透明质酸合成酶1，2，3 mRNA以及透明质酸表达的差异与空白对照组相比，重组人骨桥蛋白干预组骨桥蛋白mRNA升高(3.53±0.17)倍，骨桥蛋白siRNA干预组骨桥蛋白mRNA水平降低(0.62±0.21)倍，3组之间差异有显著性意义(P < 0.05)，见图3A。与空白对照组相比，重组人骨桥蛋白干预组骨桥蛋白表达水平升高(1.68±0.23)倍，骨桥蛋白siRNA干预组骨桥蛋白表达水平降低(0.72±0.20)倍，3组之间差异有显著性意义(P < 0.05)，见图3B。与空白对照组相比，重组人骨桥蛋白干预组透明质酸合成酶1 mRNA表达水平升高(2.32±0.23)倍，骨桥蛋白siRNA干预组透明质酸合成酶1 mRNA表达水平降低(0.51±0.05)倍，3组之间差异有显著性意义(P < 0.05)，见图3C。与空白对照组相比，重组人骨桥蛋白干预组透明质酸合成酶2 mRNA表达水平升高(3.31±0.10)倍，骨桥蛋白siRNA干预组透明质酸合成酶2 mRNA表达水平降低(0.59±0.02)倍，3组之间差异有显著性意义(P < 0.05)，见图3D。与空白对照组相比，重组人骨桥蛋白干预组透明质酸合成酶3 mRNA表达水平升高(7.28±0.31)倍，骨桥蛋白siRNA干预组透明质酸合成酶3 mRNA表达水平降低(0.37±0.06)倍，3组之间差异有显著性意义(P < 0.05)，见图3E。与空白对照组(299.7±3.00) ng/L相比，重组人骨桥蛋白干预组透明质酸表达水平升高(884.00±7.61) ng/L，骨桥蛋白siRNA干预组透明质酸表达水平降低(207.02± 4.07) ng/L，3组之间差异有显著性意义(P < 0.05)，见图3F。"

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