天津医药 ›› 2015, Vol. 43 ›› Issue (7): 792-796.doi: 10.11958/j.issn.0253-9896.2015.07.025

• 诊断技术 • 上一篇    下一篇

吖啶橙荧光染色法在循环肿瘤细胞筛查中的应用

刘敏,马富玲,韩荣,李美,刘淑芬,张淑敏   

  1. 1.天津医科大学第二医院泌尿外科
     2.天津市泌尿外科研究所

  • 收稿日期:2014-10-11 修回日期:2015-03-04 出版日期:2015-07-15 发布日期:2015-07-15
  • 通讯作者: 马富玲△ E-mail:18722569796@163.com

The application of acridine orange fluorescent staining method in circulating tumor cell

LIU Min, MA Fuling, HAN Rong, LI Mei, LIU Shufen, ZHANG Shumin   

  1. 1.Department of Urology, The Second Hospital, Tianjin Medical University, 
    2.Tianjin Institute of Urinary Surgery, Tianjin


  • Received:2014-10-11 Revised:2015-03-04 Published:2015-07-15 Online:2015-07-15
  • Contact: MA Fuling△ E-mail:18722569796@163.com

摘要: 目的:应用吖啶橙荧光(AO-F)染色法搭建循环肿瘤细胞(CTCs)筛选平台,以及该方法在肾癌患者外周血中的应用初探。方法:传代培养肾肿瘤细胞,AO-F染色后随机计数5个视野下AO-F+细胞占肿瘤细胞的比例,评估AO-F染料对肿瘤细胞的敏感性;清晨空腹抽取10例健康体检者(对照组)外周血,红细胞裂解后富集单个核细胞,与一定数量的肿瘤细胞混合,建立肾癌外周血CTCs模型,AO-F染色后计算肿瘤细胞回收率,评估本方法特异性及稳定性;选取27例转移性肾癌患者作为实验组,采用AO-F法检测两组(对照组、实验组)外周血AO-F+细胞的检出率并分析检出率与患者临床参数之间的关系。结果: 平均每个视野下AO-F+细胞占肿瘤细胞的比例为93%±3%。含500、200、100、50个细胞的悬液组肿瘤细胞回收率(%)分别为:10.2±3.8、9.2±2.3、10.8±2.6、10.5±1.9,组间差异无统计学意义(F=1.001,P>0.05)。含10个细胞的悬液组偶然可检测到AO-F+细胞。对照组无一例阳性,实验组9例检测到AO-F+细胞,检出率为33.33%;检出率与年龄、性别、肿瘤大小、病理类型、Furhman分级、转移部位及带瘤状态均没有统计学差异(P>0.05)。结论:本研究证实了AO-F染色法筛查外周血CTCs切实可行,并具有高度的特异性和可重复性。对早期预测肾癌复发转移有一定参考价值。

关键词: 吖啶橙,  显微镜检查, 荧光,  癌, 肾细胞,  循环肿瘤细胞

Abstract: Objective :To explore the screening method of circulating tumor cells (CTCs) staining with acridine orange fluorescent (AO-F) and its application in metastatic renal cell carcinoma (RCC) patients. Methods: Culture 769-P cell line and primary RCC cells,calculate the percentage of AO-F positive staining 769-P cell under 5 random sights after AO-F staining and evaluate the sensitivity of AO-F to cells. 5ml morning fasting venous blood of all controls was obtained to enrich nucleated cells. The CTCs model of renal cancer was established when known numbers of tumor cell(500、200、100、50、10)respectively were mixed with 106 nucleated cells which were used to evaluate A0-F method’s specificity and reproducibility. 27 metastatic RCC patients were collected and use AO-F staining method to detect the expression of AO-F positive cells in their blood. Analyse the correlation between expression and clinical parameters. Results: The percentage of AO-F positive staining 769-P cell was 93%±3% under 5 random sights. The recovery rate(%) of four groups (500、200、100、50) was 10.2±3.8、9.2±2.3、10.8±2.6、10.5±1.9, respectively. No significant difference was found among four groups(F=1.001,P>0.05).The group(10)could find AO-F positive staining cells occasionally. Zero case in controls was positive. 9 cases of 27 in test group were positive and the rate was 33.33%. There were no significant statistical difference between gender, ages, tumor sizes, pathological pattern, Furhman stages, metastasis of lung or not, presence of tumor or not and AO-F positive rate (P >0.05). Conclusion: It is confirmed that the method of CTCs staining with AO-F, which has high specificity and reproducibility, is feasible to detect CTC and worthy of being studied. There is a certain reference value to predict tumor recurrence and metastasis.

Key words: acridine orange,  microscopy, fluorescence, carcinoma, renal cell, circulating tumor cells