TRPV4影响软骨细胞退变的初步研究

Preliminary study of TRPV4 affects chondrocyte degeneration

目的:探讨并验证瞬时受体电位香草素受体4(transient receptor potential vanilloid 4,TRPV4)影响软骨细胞的退变。方法:取新生SD大鼠软骨细胞,甲苯胺蓝染色和阿利新蓝染色鉴定;通过白细胞介素1β(interleukin-1β,IL-1β)构建体外软骨细胞炎症模型,采用TRPV4抑制剂处理炎症条件下软骨细胞,经荧光定量PCR(real-time PCR,PCR)法检测软骨细胞中TRPV4、基质金属肽酶-13(matrix metallopeptidase 13,MMP-13)、血小板反应蛋白解整合素金属肽酶-5(A aisintegrin and metalloproteinase with thrombospondin 5,ADAMTS-5)、一氧化氮合酶2(nitric oxide synthase 2,NOS2)、Ⅱ型胶原α1(collagen,typeⅡ,alpha 1,Col2α1)和软骨蛋白聚糖(aggrecan,Acan)mRNA;通过TRPV4不同浓度过表达质粒处理原代软骨细胞,筛选最佳过表达剂量,并经RT-PCR法检测最佳TRPV4过表达剂量下软骨细胞中TRPV4、MMP-13、ADAMTS-5、NOS2、Col2α1、Acan mRNA表达情况。结果:甲苯胺蓝染色和阿利新蓝染色鉴定提取细胞为原代软骨细胞。RT-PCR显示,经TRPV4抑制剂处理后炎症条件下软骨细胞中TRPV4、MMP-13、ADAMTS-5、NOS2 mRNA的表达明显下降(P<0.05),Col2α1 mRNA、Acan mRNA的表达升高(P<0.05);经0.8 ng·μl-1 TRPV4过表达质粒处理的软骨细胞中Col2α1、Acan mRNA表达情况明显下降(P<0.05),NOS2 mRNA的表达升高(P<0.05),MMP-13、ADAMTS-5mRNA表达差异无统计学意义(P>0.05)。结论:抑制TRPV4的表达能下调软骨细胞退变相关基因表达。

Objective To explore and verify that transient receptor potential vanilloid 4(TRPV4)affects chondrocyte degeneration.Method Neonatal SD rats were selected,primary chondrocytes were extracted,and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β,and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions,and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13),a disintegrin and metalloproteinase with thrombospondin 5,(ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen,type II alpha 1(Col2α1)and aggrecan(Acan)mRNA in chondrocytes;primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid,and the optimal overexpression dose was screened.The mRNA expressions of TRPV4,MMP-13,ADAMTS-5,NOS2,Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected.Results Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4,MMP-13,ADAMTS-5,NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions.The expression of Col2α1 mRNA was significantly decreased(P<0.05),and the expression of Col2α1 mRNA was increased(P<0.05).Although there was no significant difference in the expression of Acan mRNA,the overall trend was also increasing.The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased(P<0.05),and the expression of NOS2 mRNA was increased(P<0.05),but there was no significant difference in MMP-13 and ADAMTS-5(P>0.05).Conclusion Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.