TRPC6在IL-1β诱导类风湿关节炎成纤维细胞样滑膜细胞增殖中的作用

目的:应用RNA干扰技术研究瞬时受体电位通道6(TRPC6)对IL-1β诱导的类风湿关节炎(RA)成纤维细胞样滑膜细胞(RA-FLS)增殖的影响。方法:RT-qPCR法检测RA和骨关节炎(OA)患者滑膜组织中TRPC6 mRNA的表达水平。组织块联合酶消化法培养RA-FLS。流式细胞术鉴定RA-FLS。将不同浓度(0、0.25、0.5、1、2、4、8、16μg/L)的重组人IL-1β与RA-FLS共培养36 h,CCK-8法检测细胞活力的改变;16μg/L的IL-1β作用RA-FLS不同时间(12、24、36、48、60、72 h),CCK-8法检测细胞活力的改变。特异性TRPC6-siRNA转染RA-FLS后,采用RT-qPCR和Western blotting检测沉默效率。在IL-1β存在和不存在的条件下,CCK-8法、Ed U标记法和流式细胞术检测TRPC6干扰组与对照组的细胞活力、Ed U阳性细胞比率和(G_2/M+S)期比率的差异。结果:RA患者滑膜组织中TRPC6的mRNA表达水平相对于OA患者明显增加(P0.05)。TRPC6-siRNA能显著降低RA-FLS中TRPC6 mRNA和蛋白的表达(P0.05)。IL-1β能诱导RA-FLS增殖(P0.05)。沉默TRPC6后,在IL-1β的诱导环境下,特异性干扰组RA-FLS的活力、Ed U阳性细胞比率和(G_2/M+S)期比率与空白组和对照组相比均明显降低(P0.05),而在不含IL-1β的条件下,干扰组与空白组和对照组相比差异均无统计学显著性。结论:TRPC6参与IL-1β诱导的RA-FLS增殖过程,沉默TRPC6能降低IL-1β诱导的RA-FLS增殖水平。

Effect of TRPC6 on IL-1β-induced proliferation of rheumatoid arthritis fibroblast-like synoviocytes

AIM: To investigate the effects of transient receptor potential channel 6 (TRPC6) on the proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) induced by IL-1β. METHODS: The mRNA expression of TRPC6 in synovial tissues from RA or OA patients was studied by RT-qPCR. RA-FLS were cultured by enzyme digestion and tissue adhesion methods. The method of flow cytometry was applied to identify the RA-FLS. RA-FLS were treated with different concentrations (0, 0.25, 0.5, 1, 2, 4, 8 and 16 μg/L) of IL-1β for 36 h. The cell viability was examined by CCK-8 assay. RA-FLS were incubated with IL-1β (16 μg/L) for different time (12, 24, 36, 48, 60 and 72 h), and the cell viability was measured by CCK-8 assay. The interference efficiency of TRPC6-siRNA was determined by RT-qPCR and Western blotting. After incubation in the presence or absence of IL-1β medium, the cell viability, the percentage of EdU-positive cells and the percentage of (G₂/M+S) phase were measured by CCK-8 assay, EdU labeling assay and flow cytometry, respectively. RESULTS: The mRNA expression of TRPC6 was found in synovial tissue with higher levels in RA patients than that in OA patients. TRPC6-siRNA significantly decreased the mRNA and protein expression of TRPC6 (P<0.05). When RA-FLS were treated with IL-1β, the proliferation of RA-FLS was increased (P<0.05). The differences of the cell viability, the percentage of EdU-positive cells and the (G₂/M+S) phase percentage between TRPC6-siRNA group and blank control group or NC-siRNA group were significant, in the presence of IL-1β (P<0.05). However, they were not significant in the absence of IL-1β. CONCLUSION: TRPC6 is involved in the proliferation of RA-FLS induced by IL-1β. Silencing of TRPC6 gene inhibits the growth of RA-FLS induced by IL-1β.