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

发布时间：2019-01-02 09:55

【摘要】：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。
[Abstract]:Aim: to investigate the effect of transient receptor potential channel 6 (TRPC6) on proliferation of (RA) fibroblast synovial cells (RA-FLS) induced by IL-1 尾 in rheumatoid arthritis (RA) by RNA interference technique. Methods: the expression of TRPC6 mRNA in synovium of RA and (OA) patients with osteoarthritis was detected by RT-qPCR method. Culture of RA-FLS. by tissue Block combined with enzyme digestion Identification of RA-FLS. by flow cytometry Recombinant human IL-1 尾 was co-cultured with RA-FLS for 36 h to detect the change of cell viability by CCK-8 method. The changes of cell viability were detected by 16 渭 g / L IL-1 尾 -induced RA-FLS at different time points (12 ~ 24 ~ 36 / 48 ~ (60 ~ 72) h), CCK-8). After transfection of specific TRPC6-siRNA into RA-FLS, silencing efficiency was detected by RT-qPCR and Western blotting. In the presence and absence of IL-1 尾, CCK-8, Ed U labeling and flow cytometry were used to detect the difference between TRPC6 interference group and control group in the ratio of active, Ed U positive cells and G _ 2 / M _ S phase. Results: the expression of TRPC6 mRNA in synovial tissue of RA patients was significantly higher than that in OA patients (P0.05). TRPC6-siRNA could significantly decrease the expression of TRPC6 mRNA and protein in RA-FLS (P0.05). IL-1 尾 could induce RA-FLS proliferation (P0.05). After silencing TRPC6, the ratio of active, Ed U positive cells of RA-FLS and the ratio of G _ 2 / M _ S phase of RA-FLS in the specific interference group were significantly lower than those in the blank group and the control group (P0.05). But without IL-1 尾, there was no significant difference between the interference group and the blank group and the control group. Conclusion: TRPC6 is involved in RA-FLS proliferation induced by IL-1 尾, and silencing TRPC6 can decrease RA-FLS proliferation induced by IL-1 尾.
【作者单位】：中山大学孙逸仙纪念医院骨科;广州市增城区人民医院骨科;佛山市南海区人民医院骨科;广州医科大学生理教研室;
【基金】：国家自然科学基金资助项目(No.81470205) 广州市属高校科研计划(No.2012C043)
【分类号】：R593.22

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