TRPV4通道在膝骨关节炎软骨细胞中的功能表达*

Functional expression of TRPV4 channels in chondrocytes of knee osteoarthritis*

摘要:

目的 基于瞬时感受器电位离子通道家族（TRP）香草素受体亚家族成员Ⅳ型（TRPV4）的特点，探讨其在人类软骨细胞中的功能性表达，以及对于膝骨关节炎（KOA）的意义。方法 人类KOA软骨细胞原代培养。分别用浓度为0、1、10和100?ng/ml，以及浓度为1和10?μg/ml的脂多糖（LPS）全培孵育24?h，或用浓度为1?μg/ml的LPS全培分别孵育0、4、8、12、24和48?h，qRT-PCR检测软骨细胞TRPV4的基因表达。软骨细胞分为空白组、LPS组、LPS+TRPV4抑制剂组，行实时荧光钙成像观察各组钙离子内流情况，并提取培养上清液，酶联免疫吸附试验（ELISA）检测基质金属蛋白酶MMP-1、MMP-3及MMP-13水平。结果 浓度为1、10、100?ng/ml和1?μg/ml的LPS均能增加人类软骨细胞TRPV4通道的基因表达（P?<0.05）；实时荧光钙成像分别观察20、40和60?s的荧光强度发现：LPS组荧光强度较空白组增加（P?<0.05），LPS+TRPV4抑制剂组荧光强度较LPS组降低（P?<0.05）；此外，LPS组的MMP-1、MMP-3及MMP-13水平高于空白组（P?<0.05），而LPS+TRPV4抑制剂组低于LPS组（P?<0.05）。结论 在KOA的炎症环境下，TRPV4不仅存在表达量升高，还存在功能表达增强。TRPV4通道可能通过影响MMP-1、MMP-3及MMP-13的表达水平参与KOA软骨降解的过程。

Abstract:

Objective To explore the functional expression of transient receptor potential vanilloid receptor 4 (TRPV4) in human chondrocytes and its significance for knee osteoarthritis (KOA) based on its characteristic. Methods Human KOA chondrocytes were primarily cultured. Then each culture was treated with LPS in the dose of 0?ng/ml, 1?ng/ml, 10?ng/ml, 100?ng/ml, 1?μg/ml, and 10?μg/ml for 24?h or treated with 1?μg/ml for the time of 0, 4, 8, 12, 24 and 48?h , respectively. RT-PCR was performed to detect the gene expression of TRPV4 in chondrocytes. Chondrocytes were divided into 3 groups: control group, LPS group and LPS+TRPV4 inhibitor group. The calcium influx in each group was observed by calcium imaging and the level of matrix metalloproteinase MMP-1, MMP-3 and MMP-13 were detected by ELISA. Results LPS with concentrations of 1, 10, 100?ng/ml and 1 μg/ml increased the gene expression of TRPV4 channels in human chondrocytes (P?< 0.05). Real-time fluorescence intensity observed in 20, 40 and 60 seconds by calcium imaging showed: the fluorescence intensity of LPS group was higher than that of control group (P?