Abstract：
In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett\'s oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment. The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett\'s oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells. The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma\'s genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.
摘要：
在本文中,我们研究了健康志愿者血浆的潜在基因毒性作用，以及胃食管反流病患者，Barrett食管（BO）和食管腺癌（OAC）使用食管腺癌细胞系（OE33）和淋巴母细胞系（TK6）。TK6和OE33细胞均用血浆处理（10％体积，在4小时（用于微核（Mn）测定和侵袭测定）和24小时（用于细胞周期研究）的不同时间点替换胎牛血清（FBS）或马血清（HS））。血浆对DNA损伤水平的诱导作用，通过微核试验研究了细胞活力和细胞周期，分别为胞质分裂阻滞增殖指数(CBPI)和流式细胞术。还通过酶联免疫吸附测定（ELISA）分析了TK6细胞上清液中IL-8和OE33细胞中IFN-β的表达。最后,我们对血浆处理后OE33细胞的细胞侵袭进行了评估.微核试验的结果证实了一些参与者通过TK6细胞中DNA损伤的增加直接血浆治疗的遗传毒性。相反，一些个体患者血浆样品降低了TK6细胞Mn频率的背景水平，以抗基因毒性的方式。在TK6细胞中，（平均）巴雷特食管患者的血浆样本比健康志愿者诱导更高的微核水平（p=0.0019）。当使用血浆与血清在体外处理细胞时,Mn诱导几乎没有差异。细胞周期结果表明，通过降低S期细胞比例，直接等离子体处理在24h对OE33细胞有显著影响(BOp=0.0182，OACp=0.0320)，而血浆暴露对TK6细胞周期的影响较小。还观察到OE33细胞的侵袭不受OE33细胞的血浆处理的影响。在剂量依赖性物质中添加N-乙酰半胱氨酸NAC并不改变TK6细胞中Mn的形成,这表明活性氧（ROS）不是血浆遗传毒性的根本原因。TK6细胞中的IL-8浓度和OE33细胞中的IFN-β浓度在用OAC衍生的血浆处理的细胞中显著高于未处理的阴性对照。总的来说，我们的结果表明,血浆特异性效应是可检测的,这有助于我们更好地了解正在开发的血液生物标志物生物学的一些重要方面.
