Molecular Cytology Core Facility, Memorial Sloan-Kettering Cancer, New York, NY, USA.
Cytotechnology. 2014 Mar;66(2):259-73. doi: 10.1007/s10616-013-9565-3. Epub 2013 Sep 25.
Radiofrequency (RF) ablation (RFA) is a minimally invasive treatment for colorectal-cancer liver metastases (CLM) in selected nonsurgical patients. Unlike surgical resection, RFA is not followed by routine pathological examination of the target tumor and the surrounding liver tissue. The aim of this study was the evaluation of apoptotic events after RFA. Specifically, we evaluated YO-PRO-1 (YP1), a green fluorescent DNA marker for cells with compromised plasma membrane, as a potential, early marker of cell death. YP1 was applied on liver tissue adherent on the RF electrode used for CLM ablation, as well as on biopsy samples from the center and the margin of the ablation zone as depicted by dynamic CT immediately after RFA. Normal pig and mouse liver tissues were used for comparison. The same samples were also immunostained for fragmented DNA (TUNEL assay) and for active mitochondria (anti-OxPhos antibody). YP1 was also used simultaneously with propidium iodine (PI) to stain mouse liver and samples from ablated CLM. Following RFA of human CLM, more than 90 % of cells were positive for YP1. In nonablated, dissected pig and mouse liver however, we found similar YP1 signals (93.1 % and 65 %, respectively). In samples of intact mouse liver parenchyma, there was a significantly smaller proportion of YP1 positive cells (22.7 %). YP1 and PI staining was similar for ablated CLM. However in dissected normal mouse liver there was initial YP1 positivity and complete absence of the PI signal and only later there was PI signal.
This is the first time that YP1 was applied in liver parenchymal tissue (rather than cell culture). The results suggest that YP1 is a very sensitive marker of early cellular events reflecting an early and widespread plasma membrane injury that allows YP1 penetration into the cells.
射频消融（RFA）是一种微创治疗方法，适用于选定的非手术患者的结直肠癌肝转移（CLM）。与手术切除不同，RFA 后不会对目标肿瘤和周围肝组织进行常规病理检查。本研究的目的是评估 RFA 后的细胞凋亡事件。具体来说，我们评估了 YO-PRO-1（YP1），一种绿色荧光 DNA 标记物，用于检测细胞膜受损的细胞，作为细胞死亡的早期潜在标志物。YP1 应用于紧贴用于 CLM 消融的 RF 电极的肝组织上，以及在 RFA 后立即通过动态 CT 描绘的消融区域的中心和边缘的活检样本上。正常猪和鼠肝组织用于比较。同一样本也用断裂 DNA（TUNEL 测定）和活性线粒体（抗 OxPhos 抗体）进行免疫染色。YP1 也同时与碘化丙啶（PI）一起用于染色鼠肝和消融的 CLM 样本。在 RFA 消融人 CLM 后，超过 90%的细胞呈 YP1 阳性。然而，在未经消融的分离猪和鼠肝中，我们发现了类似的 YP1 信号（分别为 93.1%和 65%）。在完整的鼠肝实质样本中，YP1 阳性细胞的比例明显较小（22.7%）。YP1 和 PI 染色在消融的 CLM 中相似。然而，在分离的正常鼠肝中，最初存在 YP1 阳性，完全没有 PI 信号，只有后来才有 PI 信号。
这是首次将 YP1 应用于肝实质组织（而不是细胞培养物）。结果表明，YP1 是早期细胞事件的非常敏感标志物，反映了早期和广泛的细胞膜损伤，允许 YP1 渗透到细胞中。
