给编辑:
胸膜液(PF)中的细胞因子水平已成为许多报道的主题,重点是其病因,诊断和预后角色。此外,细胞因子水平在胸膜疾病的翻译研究中很重要1。但是,这些研究的数据基于对融化后分析的储存的PF样品中细胞因子的单个“一次性”测量。细胞因子浓度是细胞因子的全身性和肺过滤的微妙平衡,并从局部(间皮和炎症和/或癌症)释放2。Reproducibility of PF cytokine measurements in humans has not previously been established. Our study aimed to: 1) determine short- and long-term changes of PF cytokine concentrations in patients with recurrent/persistent pleural effusions; and 2) to examine the influence of freeze–thaw cycles on cytokine levels.
总共获得了30名需要留置胸膜导管(n = 19)或重复的胸膜攻击(n = 11)的患者的137个串行PF样品,以治疗恶性(n = 24)或良性(n = 6)经常性液相。。这些患者中有26例是针对使用PF间皮素的单独研究的一部分收集的PF3。附加的样本收集的年代even patients (malignant, n = 5; benign, n = 2) to assess the effects of storage and freeze–thaw cycles. Short-term reproducibility was assessed between “paired” PF samples taken from patients within any 10-day period. Multiple paired samples were included from patients who had repeated sampling. PF samples were collected and centrifuged as previously published. Supernatants were stored at -80°C until assayed. Vascular endothelial growth factor (VEGF), interleukin (IL)-8, tumour necrosis factor (TNF)-α, macrophage chemoattractant protein (MCP)-1, and transforming growth factor (TGF)-β1 and TGF-β2 concentrations were measured using commercially available ELISA kits (PeproTech EC Ltd, London, UK). Total TGF-β levels were measured by activating the PF samples with HCl, followed by neutralisation with hydroxyethyl piperazine ethane sulphonic acid-NaOH before ELISA assaying, as recommended by the manufacturer. Analyses were repeated on all paired serial samples showing a more than two-fold change for confirmation.
在10天内采用的配对样品中细胞因子测量的短期可重复性非常好(表1⇓)。No significant changes were observed in the cytokines tested (all p≥0.05). Likewise, PF cytokine concentrations remained stable during each patient’s disease course. There was no significant change over time in all cytokines tested from samples obtained at intervals 1–242 days apart (median 45 days). Cytokine levels correlated significantly with each other, in particular, the inter-relationships between VEGF, MCP-1, IL-8 and TGF-β1 were highly significant (all p<0.001). No obvious differences were noted between malignant and benign samples (data not shown).
To assess the stability of cytokines during the freeze–thaw cycle, PF samples were immediately placed into multiple aliquots after centrifugation and stored at -80°C. One aliquot was thawed (to 0°C on ice or to room temperature for 2 h) and frozen again at -80°C for ≥6 h. For each PF sample, separate aliquots were frozen then thawed for a total of one, two, three and eight times and analysed simultaneously. The absolute percentage change in cytokine levels (eight cycles相对基线)如下(平均值±sd)。VEGF:6.76±6.98%(p = 0.59);MCP-1:25.33±27.84%(p = 0.66);TNF-α:18.05±20.45%(p = 0.63);TGF-β1:11.6±13.75%(p = 0.35);TGF-β2:74.93±88.48%(p = 0.66);和IL-8:10.08±7.23%(p = 0.54)。在所有情况下,变化的方向都不同,没有任何建议是通过重复冻结周期的总体降低或值增加(图1⇓)。同样,在除霜到0°C的样品中未观察到显着变化(数据未显示)。
这项研究首次表明,PF中常见测试的细胞因子的测量值得高度重现,并且在实际处理PF样品时所必需的重复冻结 - 融化周期之后是稳定的。这为涉及PF细胞因子定量及其潜在临床应用的研究提供了至关重要的验证。此外,这些细胞因子的测量并不取决于疾病过程中的时间,这为这些细胞因子的诊断使用的研究增加了力量。
It is intriguing that the cytokine levels are remarkably stable in the pleural cavity despite the many potential influencing factors,例如cell release and metabolism, serum extravasation and systemic reabsorption. It is logical then to assume they are tightly regulated, even in the disease states, for important biological reasons and testify to the current belief that these cytokines hold vital roles in disease biology.
我们的研究有局限性。研究的大多数样品都是恶性胸腔积液,反映出它们是复发积液的最常见原因。尽管我们样品中显示的细胞因子的可重复性和稳定性可能会扩展到其他病因的复发或持续积液,以及其他细胞因子,但这需要确认。
Support statement
Y.C.G. Lee received funding from the Medical Research Council (London, UK), the National Health and Medical Research Council (Canberra, Australia) and received the Dept of Health Clinical Senior Lecturer Award (UK Clinical Research Collaboration, London, UK). The Instituto de Salud Carlos III (FIS CM07/00020) provided funding to S. Bielsa. The British Lung Foundation provided funding to H.E. Davies, R.J.O. Davies and Y.C.G. Lee, and funding was given to R.J.O. Davies from the Oxford National Institute of Health Research Biomedical Research Centre Programme (Oxford, UK).
Statement of interest
None declared.
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