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| 膝关节骨性关节炎中WISP-1调控机制的研究 |
| Study of WISP-1 signal regulation mechanism in knee joint osteoarthritis |
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| DOI： |
| 中文关键词: 膝关节骨性关节炎 软骨细胞 WISP-1 ；信号传导；细胞外基质 |
| 英文关键词:Osteoarthritis Cartilage WISP-- Signaling Extracellular matrix |
| 基金项目:国家自然科学基金项目（81071499) |
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| 中文摘要: |
| 目的使用膝关节骨性关节炎（OA)临床组织标本与人软骨细胞株(NHAC).以分子生物学手法探讨WISP4在骨关节 软骨基质合成和分泌中的调控机理，明确其在骨性关节炎中的作用。方法NHAC细胞以1. 4 x 106/mL密度播种于6孔板 (直径3.5 cm)后培养48 h，于含有0.2%牛血清白蛋白（BSA)的DF中继续培养12 h，使细胞处于静止状态。向含0.02% BSA 的 DF 培养基中加人 10、50 和 100 ng/mL 的 Wnt 刺激细胞 6 h。对照 siRNA 与 P~catenin siRNA，使用 EntranserTM~R (Engreen)转染试剂转染至NHAC。转染24 h后使用PBS清洗细胞两次。向含0.02% BSA的DF培养基中加人50 ngjnL的 Wnt刺激细胞。向NHAC负荷不同剂量WISP4，24 h后观察MMPs和ADAMTS4。利用总RNA提取试剂盒提取组织标本和细 胞RNA，反转录产物cDNA作为PCR扩增模板进行定量PCR;使用蛋白提取试剂盒提取蛋白。结果在膝关节组织标本中发 现，在骨性关节炎关节软骨中WISP~1、Wnt和b |
| 英文摘要: |
| Objective To explore the regulation mechanism of WISP-L in the formation and secretion of the joint cartilage matrix by using human osteoarthritis (OA) tissue sample and human chondrocyte cell line (NHAC)，and to confirm the role of WISP-- in the joint degenerative disease. Methods NHAC cells were seeded in the 6-well plates (3. 5cm in diameter) with a density of 1. 4 x 106/ml and cultured for 48 h. Then they were cultured in DF with 0. 2% BSA for further 12 h. When the cells were stable，they were treated with 10，50，and 100 ng/ml of Wnt for 6h，respectively. Control siRNA and p-catenin siRNA were transfected into NHAC using the EntraserTM-R kit (Engreen). The cells were washed with PBS twice after 24 h transfection. 50 ng/ml of Wnt was additioned to DF with 0.02% BSA to stimulate the cells. Different concentrations of WISP-1 were loaded. MMPs and ADAMTS4 were observed after 24h. Total RNA was extracted from the cells using a commercial kit. Quantitative RT-PCR was performed. Protein was extracted with an extraction kit. Results Protein and mRNA expression of WISP--，Wnt，and p-catenin in human OA cartilage were higher than those in control cartilage. WISP-- expression increased significantly in a dose dependent manner under the Wnt stimulation. After knocking down the endogenous p-catenin，the expression of WISP-- was significantly down-regulated. The gene expressions of MMP-3，--，-3，and ADAMTS4 were induced dose-dependently by WISP--. Conclusion WISP-- is related to the development of OA. In the cartilage cells WISP-- can be induced by Wnt through p-catenin signal pathway. |
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