吖啶橙荧光染色法在循环肿瘤细胞筛查中的应用

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

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,并将其应用于肾癌患者外周血CTCs筛查。方法选择转移性肾细胞癌患者27例(研究组),用不同浓度的血清培养肾癌原代肿瘤细胞和细胞系769-P,分别制备涂片于荧光显微镜下,观察细胞形态及着色情况是否一致;随机计数5个视野下AO-F阳性(+)细胞占769-P细胞的比例,评估AO-F染料对肿瘤细胞的敏感性;清晨抽取10例健康体检者(对照组)空腹外周血,红细胞裂解后富集单个核细胞,配制含1×106个细胞的悬液;取对数生长期肾肿瘤细胞,配制成每管分别含500、200、100、50、10个肿瘤细胞的悬液,与1×106个单个核细胞混合,建立肾癌外周血CTCs模型,AO-F染色后计算肿瘤细胞回收率;另计算2组外周血AO-F+细胞的检出率,分析其与患者临床参数之间的关系。结果肾癌原代肿瘤细胞和细胞系769-P均呈现出鲜明色彩,两者具有相似的形态学特征,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(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,研究组9例检测到AO-F+细胞,检出率为33.33%;检出率在不同年龄、性别、肿瘤大小、病理类型、Furhman分级、转移部位及带瘤状态间差异均无统计学意义(P>0.05)。结论 AO-F染色法筛查肾癌患者外周血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。

Objective To establish the screening platform of circulating tumor cells（CTCs） using acridine orange fluo-rescent（AO-F） dyeing method, and to apply it in the screening of peripheral blood CTCs in patients with kidney cancer.Methods Twenty-seven patients with metastatic renal cell carcinoma was included in this study. Primitive tumor cells andkidney cancer cell line 769-P were cultured with different concentrations of fetal bovine serum. Smears were prepared andobserved under fluorescence microscopy. The percentage of AO-F positive staining of 769-P cells under 5 random sightswas calculated. The sensitivity of AO-F staining to cells was evaluated. The 5 m L morning fasting venous blood was obtainedfrom 10 subjects with healthy check-up. The 1×106cell suspension was prepared. The logarithmic phase of renal tumor cellswas used to prepare tube containing 500, 200, 100, 50 and 10 tumor cell suspension, which were mixed with 1×106nucleatedcells to establish CTCs model of renal cancer. AO-F staining method was used to detect the expression of AO-F positivecells. The correlation between expression of AO-F positive cells and clinical parameters was analyzed. Results The prima-ry cells and cell line 769-P showed similar bright color and morphological characteristics. The percentage of AO-F positivestaining in 769-P cells was 93%±3% under 5 random sights. The recovery rates（%） of four groups（500, 200, 100 and 50 tu-mor cell suspension） were 10.2±3.8, 9.2±2.3, 10.8±2.6 and 10.5±1.9, respectively. There were no significant differences inrecovery rates between four groups（P0.05）. The group of 10 tumor cell suspension could find AO-F positive staining cellsoccasionally. Zero case was positive in controls. Nine of 27 patients were positive and the rate was 33.33%. There were nosignificant statistical differences in AO- F positive rates between gender, age, tumor size, pathological pattern, Furhmanstage, metastasis of lung and presence of tumor（P〉0.05）. Conclusion It is confirmed that the method of CTCs stainingwith AO-F, which has high specificity and reproducibility, is feasible to detect CTCs and worthy of being studied. There is acertain reference value to predict tumor recurrence and metastasis.