人工关节磨屑颗粒诱导巨噬细胞分泌细胞因子与依那西普干预的影响

Interventional effect of etanercept on cytokine secretion from debris-induced macrophages

目的:已证实肿瘤坏死因子α在人工关节无菌性松动过程中发挥重要作用,依那西普为肿瘤坏死因子α拮抗剂,拟验证采用依那西普预防人工关节钛颗粒刺激巨噬细胞分泌肿瘤坏死因子α等细胞因子导致无菌性松动的可能性。方法:实验于2007-03/07在宁夏医学院生殖与遗传实验室完成(宁夏省部级重点实验室)。①主要试剂与药物:依那西普(Enbrel,Amgenand Wyeth),钛颗粒(美国ZIM-MER公司提供,微粒直径为3～5μm)。②小鼠腹腔巨噬细胞的分离、培养:麻醉后处死12只6～8周的清洁级BALB/C小鼠,腹腔注入无血清RPMI1640培养液5mL,3～5min后,无菌条件下打开小鼠腹腔,吸取腹腔液,离心洗涤后显微镜下计数,调整细胞至3×109/L,均匀加入24孔培养板中,置于37℃、体积分数为0.05的CO2培养箱中培养。12h后更换培养液,去掉未贴壁的细胞,得到巨噬细胞。实验过程中对动物处置符合动物伦理学要求。③评估指标:24h后将培养细胞分为5组,每组有8个平行孔:单纯细胞组,1×1012/L钛颗粒组,1×1012/L钛颗粒+10μg/L依那西普组,1×1012/L钛颗粒+100μg/L依那西普组,1×1012/L钛颗粒+1000μg/L依那西普组。继续培养18h后,用酶联免疫法检测上述各组细胞上清液中肿瘤坏死因子α、白细胞介素1、白细胞介素6的浓度。结果:1×1012/L钛颗粒组细胞培养上清液肿瘤坏死因子α、白细胞介素6、白细胞介素1浓度明显高于单纯细胞组、1×1012/L钛颗粒+100,1000μg/L依那西普组(P<0.001)。1000μg/L依那西普组低于10μg/L依那西普组,差异有显著性(P<0.001)。结论:依那西普呈剂量依赖性的有效抑制磨屑颗粒诱导的巨噬细胞分泌细胞因子,有望成为预防人工关节无菌性松动的药物。

AIM：It has been demonstrated that tumor necrosis factor alpha （TNF-α） plays an important pathogenetic role in the aseptic loosening of artificial prosthesis. This study explored the possibility for etanercept, anti-TNF-α agents, to prevent the aseptic loosening of prosthesis caused by TNF-α. METHODS： The experiment was performed at the Generation and Heredity Laboratory of Ningxia Medical College （Ningxia Key Laboratory） from March to July 2007. ①The main agent and drugs involved etanercept （Enbrel, Amgen and Wyeth） and titanium particles （ZIM-MER Co, American;3-5μm in diameter）. ②Twelve BALB/C mice aged 6-8 weeks of clean grade were executed under anesthesia, and 5 mL RPMI1640 serum-free culture solution was injected into the abdominal cavity. After 3-5minutes, the abdominal cavity was opened to imbibe peritoneal fluid in sterile condition, then the cell count was performed after centrifugalization and ablution by microscope. After the density of cells was modulated to 3×10^9/L, the cells were evenly put in 24-well culture plate and cultivated in 0.05 volume fraction CO2 incubator of at 37 ℃ for 12 hours. The culture solution was replaced to remove the unattached cells and obtain macrophages. The disposition on experimental animal conformed to the animal ethical standards. ③Twenty-four hours later, the cultured cells were divided into 5 groups with 8 wells in each group： simple cells group, 1×10^12 /L titanium particles group, 1×10^12 /L titanium particles ＋ etanercept （10 μg/L, ） group, 1×10^12 /L titanium particles ＋ etanercept （100 μg/L） group, and 1×10^12 /L titanium particles ＋ etanercept （1 000 μg/L） group. After cultivating for 18 hours, the concentrations of TNF-α, interleukin-1 （IL-1） and IL-6 in the culture supernatants of each group were detected by enzyme linked immunosorbent assay （ELISA）. RESULTS： The concentrations of TNF-α, IL-1 and IL-6 in culture supernatants of 1×10^12 /L titanium particles group were significantly higher than those in simple cells group, 1×10^12 /L titanium particles ＋ etanercept （100 μg/L） and （1 000 μg/L） groups （P 〈 0.001）. Compared with 1×1012 /L titanium particles ＋ etanercept group （10 μg/L）, they were decreased significantly in 1×10^12 /L titanium particles ＋ etanercept （1000 μg/L） group （P 〈 0.001）. CONCLUSION： Etanercept can significantly inhibit the cytokines secreted by debris-induced macrophages in a dose-dependent manner. It is promising to be a therapeutic candidate for the prevention of aseptic loosening of prosthesis.