吖啶橙荧光染色法在循环肿瘤细胞筛查中的应用
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,...
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| Published in | 天津医药 Vol. 43; no. 7; pp. 792 - 795 |
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| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
天津医科大学第二医院泌尿外科,天津市泌尿外科研究所 邮编,300211
2015
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| Abstract | 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。 |
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| AbstractList | 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。 R737.11%R446-39; 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。 |
| Abstract_FL | 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 and kidney cancer cell line 769-P were cultured with different concentrations of fetal bovine serum. Smears were prepared and observed under fluorescence microscopy. The percentage of AO-F positive staining of 769-P cells under 5 random sights was calculated. The sensitivity of AO-F staining to cells was evaluated. The 5 mL morning fasting venous blood was obtained from 10 subjects with healthy check-up. The 1×106 cell suspension was prepared. The logarithmic phase of renal tumor cells was used to prepare tube containing 500, 200, 100, 50 and 10 tumor cell suspension, which were mixed with 1×106 nucleated cells to establish CTCs model of renal cancer. AO-F staining method was used to detect the expression of AO-F positive cells. 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 positive staining 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 in recovery rates between four groups (P>0.05). The group of 10 tumor cell suspension could find AO-F positive staining cells occasionally. Zero case was positive in controls. Nine of 27 patients were positive and the rate was 33.33%. There were no significant statistical differences in AO-F positive rates between gender, age, tumor size, pathological pattern, Furhman stage, metastasis of lung and presence of tumor (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 CTCs and worthy of being studied. There is a certain reference value to predict tumor recurrence and metastasis. |
| Author | 刘敏 马富玲 韩荣 李美 刘淑芬 张淑敏 |
| AuthorAffiliation | 天津医科大学第二医院泌尿外科,天津市泌尿外科研究所,300211 |
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| Author_FL | LIU Shufen LIU Min LI Mei HAN Rong ZHANG Shumin MA Fuling |
| Author_FL_xml | – sequence: 1 fullname: LIU Min – sequence: 2 fullname: MA Fuling – sequence: 3 fullname: HAN Rong – sequence: 4 fullname: LI Mei – sequence: 5 fullname: LIU Shufen – sequence: 6 fullname: ZHANG Shumin |
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| Keywords | carcinoma,renal cell 显微镜检查,荧光 microscopy,fluorescence circulating tumor cells 癌,肾细胞 acridine orange 吖啶橙 循环肿瘤细胞 |
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| Notes | 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 |
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| SubjectTerms | 吖啶橙 循环肿瘤细胞 显微镜检查 癌 肾细胞 荧光 |
| Title | 吖啶橙荧光染色法在循环肿瘤细胞筛查中的应用 |
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