Effects of Tumor Necrosis Factor Alpha on the Expression of Programmed Cell Death Factor 5 in Arthritis
Effects of Tumor Necrosis Factor Alpha on the Expression of Programmed Cell Death Factor 5 in Arthritis
复制标题
肿瘤坏死因子α对关节炎中程序性细胞死亡因子5表达的影响
DOI：
10.1111/os.12497
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发表时间：
2019-07-08
影响因子：
2.1
通讯作者：
Guan, Zhen-peng
中科院分区：
文献类型：
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作者：
Zhang, Ke-shi;Wang, Jun-feng;Guan, Zhen-peng
目的探讨肿瘤坏死因子-α（TNF-α）对炎症微环境中成纤维样滑膜细胞（FLS）增殖及程序性细胞死亡因子5（PDCD 5）表达的影响，进一步了解TNF-α促进滑膜细胞增殖和软骨细胞凋亡的作用机制。方法21例骨性关节炎患者和12例健康股骨颈骨折患者在关节置换术中获得关节滑架标本。采用ImagePro Plus软件检测PDCD 5的平均选择密度（MOD），通过免疫荧光分析评价PDCD 5的表达。实时荧光定量PCR检测滑膜中PDCD 5和TNF-α的表达。将来源于类风湿性关节炎患者的FLS细胞在体外培养并与不同浓度的TNF-α孵育。采用CCK-8法检测不同浓度的TNF-α对FLS细胞增殖的影响，评价细胞增殖率。在不存在或存在不同浓度的重组人TNF-α的情况下孵育后，分离FLS细胞用于检测PDCD 5蛋白和PDCD 5基因。Western-blot检测PDCD 5蛋白的表达，实时荧光定量PCR检测PDCD 5基因的转录。结果骨关节炎软骨组织中PDCD 5和TNF-α的MOD均较对照组明显增高，滑膜中PDCD 5和TNF-α mRNA的表达与对照组相比呈显著正相关。当TNF-α浓度为1 ng/mL时，细胞增殖率与对照组无显著差异（P = 0.592），而当TNF-α的浓度为5、10、15或20 ng/mL时，FLS细胞的增殖显著促进，增殖促进率为35.64% +/-6.96%，分别为48.72% ± 7.69%、45.60% ± 8.85%和39.32% ± 6.18%（P < 0.01）。10和15 ng/mL的TNF-α作用FLS细胞24 h后，PDCD 5基因转录水平明显下调，分别为80.44% ± 4.07%和84.30% ± 5.48%（P < 0.05）。当TNF-α浓度为1、5、20 ng/mL时，FLS细胞中PDCD 5 mRNA的转录水平与对照组相比差异无统计学意义（P > 0.05）。当TNF-α浓度为10 ng/mL时，FLS细胞中PDCD 5蛋白的表达才显著下调（P < 0.01），而与对照组相比，FLS细胞中PDCD 5蛋白的表达无显著差异（P > 0.05）。结论PDCD 5和TNF-α在骨关节炎软骨和滑膜中的表达明显高于正常组织，TNF-α可促进类风湿关节炎患者FLS细胞增殖，抑制PDCD 5的表达。PDCD 5可能参与TNF-α刺激下滑膜细胞的异常增殖和软骨细胞的变性。
Objective To investigate the effect of tumor necrosis factor alpha (TNF-alpha) on the proliferation of fibroblast-like synoviocytes (FLS) and the expression of programmed cell death factor 5 (PDCD5) in an inflammatory microenvironment, for the further understanding of the mechanism of action of TNF-alpha in promoting the proliferation of synovial cells and the apoptosis of the chondrocytes. Methods Articular carriage specimens were obtained from 21 cases with osteoarthritis and 12 cases with femoral neck fractures as healthy controls during arthroplasties. The expression of PDCD5 was evaluated by immunofluorescence analyzed by mean option density (MOD) detected using the software ImagePro Plus. Real-time PCR was performed to evaluate the transcriptions of PDCD5 and TNF-alpha in synovium. FLS cells derived from rheumatoid arthritis patients were cultured in vitro and incubated with different concentrations of TNF-alpha. The effects of TNF-alpha at different concentrations on the proliferation of FLS cells were detected by Cell Counting Kit-8 (CCK-8) assay to evaluate the cell proliferation rate. After incubation with the absence or presence of recombinant human TNF-alpha at different concentrations, the FLS cells were isolated for detection of PDCD5 protein and PDCD5 gene. The expression of PDCD5 protein was detected by western-blot and the transcription of PDCD5 gene from the cells was detected by real-time quantitative PCR. Results The MOD of PDCD5 as well as TNF-alpha of osteoarthritis cartilage sections were significantly increased compared with those of the controls, and in synovium there was a positive correlation between transcriptions of their mRNA. When the concentration of TNF-alpha was 1 ng/mL, the cell proliferation rate was not significantly different from that of the control group (P = 0.592), while the proliferation of FLS cells was significantly promoted when the concentration of TNF-alpha was 5, 10, 15, or 20 ng/mL, and the proliferation-promoting rates were 35.64% +/- 6.96%, 48.72% +/- 7.69%, 45.60% +/- 8.85%, and 39.32% +/- 6.18%, respectively (P < 0.01). The transcription of PDCD5 gene was significantly downregulated, which was 80.44% +/- 4.07% and 84.30% +/- 5.48%, respectively (P < 0.05), in the FLS cells incubated with TNF-alpha at the concentration of 10 and 15 ng/mL for 24 h. When the concentration of TNF-alpha was 1, 5, or 20 ng/mL, the transcription of PDCD5 mRNA in FLS cells was not significantly different from that in the control group (P > 0.05). The expression of PDCD5 protein was only significantly downregulated when the concentration of TNF-alpha was 10 ng/mL (P < 0.01), while the expression of PDCD5 protein in FLS cells was not significantly different from that in the control group (P > 0.05). Conclusion The expression of PDCD5 as well as TNF-alpha in osteoarthritis cartilage and synovium was significantly higher than in healthy tissues, and TNF-alpha can promote the proliferation of FLS cells in patients with rheumatoid arthritis, and inhibit the expression of PDCD5. PDCD5 may be involved in the abnormal proliferation of synoviocytes and the degeneration of chondrocytes stimulated by TNF-alpha.