沉默脂联素受体1对脂多糖诱导的类风湿关节炎滑膜成纤维细胞MH7A增殖凋亡的影响

发布时间：2018-03-18 07:36

本文选题：脂联素受体　切入点：类风湿关节炎　出处：《南京医科大学学报(自然科学版)》2017年09期 　论文类型：期刊论文

【摘要】：目的 :观察脂联素受体1(adiponectin receptor1,AdipoR1)沉默对脂多糖(lipopolysaccharide,LPS)诱导的人类风湿关节炎滑膜成纤维细胞(MH7A)增殖、凋亡的影响。方法:(1)利用免疫荧光、Western blot鉴定并验证shRNA技术对构建的AdipoR1沉默MH7A(sh-AdipoR1组)细胞的干扰效率;(2)分别用CCK8试剂盒、流式细胞仪检测LPS(100 ng/m L)对sh-AdipoR1组及空载病毒对照(sh-NC组)细胞增殖及凋亡的影响;(3)利用实时定量聚合酶链反应法(real-time polymerase chain reaction,realtime PCR)检测LPS对sh-AdipoR1及sh-NC组细胞中凋亡相关基因BCL-2、BCL-XL、BAX、BAK表达的影响。同时设置无LPS刺激的sh-NC、sh-AdipoR1组作为对照。结果:(1)荧光显微镜下可见sh-AdipoR1组细胞荧光蛋白表达效率趋近于100%,Western blot检测结果显示:与sh-NC组相比,sh-AdipoR1组AdipoR1蛋白的表达显著下降(P0.05),表明AdipoR1沉默的细胞构建成功;(2)无LPS刺激情况下,sh-NC组与sh-AdipoR1组细胞增殖速率及凋亡细胞比例无明显差异;LPS刺激可显著增加sh-NC组和sh-AdipoR1组细胞相对增殖速率及凋亡细胞比例(P0.05);在LPS作用24、48 h,sh-AdipoR1组细胞增殖速率均显著低于sh-NC组(P0.05),而sh-AdipoR1组细胞凋亡率显著高于sh-NC组(P0.05);(3)real-time PCR结果显示,无LPS刺激情况下,sh-NC组与sh-AdipoR1组抑制凋亡基因BCL-2、BCL-XL与促进凋亡基因BAK、BAX的相对表达量均无显著差异(P0.05);LPS刺激可降低抑凋亡基因BCL-2、BCL-XL表达,增加促凋亡基因BAX、BAK表达(P0.05);在LPS诱导下与sh-NC组相比,sh-AdipoR1组抑制凋亡基因BCL-2、BCL-XL表达下降,促凋亡基因BAX、BAK表达显著升高(P0.05)。结论:AdipoR1基因的沉默可抑制LPS诱导的MH7A细胞增殖,促进细胞凋亡,并提示AdipoR1相关信号通路可能在类风湿关节炎发生发展中起到重要作用,阻断该途径可有效阻断LPS诱导的MH7A细胞炎症反应。
[Abstract]:Aim: to observe the effect of adiponectin receptor 1 adipoR1 (AdipoR1) silencing on the proliferation of human rheumatoid arthritis synovial fibroblasts (MH7A) induced by lipopolysaccharide (LPS). Methods the interference efficiency of shRNA on AdipoR1 silenced MH7A(sh-AdipoR1 cells was identified and verified by Western blot. Methods CCK8 kit was used to identify and verify the interference efficiency of shRNA on AdipoR1 silencing MH7A(sh-AdipoR1 cells. Effect of LPS(100 ng/m L) on cell proliferation and apoptosis in sh-AdipoR1 group and non-loaded virus control sh-NC group by flow cytometry) real-time polymerase chain reactionation realtime PCR was used to detect the apoptosis related gene BCL-2 and BCL-XLBAXBAK in sh-AdipoR1 and sh-NC cells by real-time polymerase chain reactiontime PCR. At the same time, the expression efficiency of fluorescent protein in sh-AdipoR1 group was closer than that in sh-NC group. The results showed that compared with sh-NC group, the expression of AdipoR1 protein in sh-NCncncdipoR1 group was higher than that in sh-NC group. There was no significant difference in cell proliferation rate and ratio of apoptotic cells between sh-AdipoR1 group and AdipoR1 group without LPS stimulation. LPS-induced cell proliferation rate increased significantly in sh-NC group and sh-AdipoR1 group. The percentage of apoptotic cells in the LPS group was significantly lower than that in the sh-NC group (P 0.05), and the apoptosis rate in the sh-AdipoR1 group was significantly higher than that in the sh-NC group. The results of real-time PCR showed that the cell proliferation rate in the LPS group was significantly lower than that in the sh-NC group, and that in the sh-AdipoR1 group was significantly higher than that in the sh-NC group, and that in the sh-AdipoR1 group was significantly higher than that in the sh-NC group. There was no significant difference in the relative expression of BCL-2nBCL-XL between the control group and the sh-AdipoR1 group without LPS stimulation. P0.05LPs could decrease the expression of BCL-2nBCL-XL. The expression of BCL-XL, BCL-2nBCL-XL and the expression of BCL-XL in BCL-2nBCL-XL in LPS group were significantly decreased compared with those in sh-NC group. Conclusion the silencing of Bax-AdipoR1 gene can inhibit the proliferation of MH7A cells induced by LPS and promote cell apoptosis. It is suggested that AdipoR1 related signaling pathway may play an important role in the pathogenesis and development of rheumatoid arthritis. Blocking this pathway can effectively block the inflammatory response of MH7A cells induced by LPS.
【作者单位】：南京医科大学第一附属医院风湿免疫科;
【基金】：国家自然科学基金(81671615) 重点病种规范化诊疗研究(BL20130134) 江苏省科技厅基础研究计划(BK2012875)
【分类号】：R593.22

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