近红外七甲川花菁染料IR-80用于鉴别甲状腺良恶性结节的实验研究

发布时间：2018-03-01 16:10

本文关键词： NIR IR-80 良恶性细胞甲状腺癌 FNA　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:本研究首先根据乳腺正常上皮细胞株(MCF-10A)和乳腺癌细胞株(MCF-7和VDA-MB231)对近红外(NIR)七甲川花菁染料IR-80不同摄取量导致的荧光强度差异性,筛选出染料浓度和拍摄参数等能够鉴别良恶性细胞最佳实验条件;检测IR-80染料孵育后甲状腺癌和良性甲状腺结节荧光强度,与传统甲状腺穿刺细胞学诊断结果比较,评估IR-80染色方法用于辅助鉴别甲状腺结节细针穿刺(FNA)标本良恶性可能性。方法:细胞系试验:通过倍比稀释配制四种浓度的IR-80染料应用液(5μM、2.5μM、1.25μM和0.625μM),分别与人乳腺癌细胞株MCF-7、MDA-MB231和人正常乳腺上皮细胞株MCF-10A共同孵育,应用荧光显微镜在NIR通道下(700-900nm)观察每种细胞在0.5s、1s和2s曝光时间的荧光强度。然后用Image J图像处理软件计算平均荧光强度,分析NIR染料IR-80对肿瘤细胞的优先聚集作用,筛选出最佳实验条件能够区别良恶性细胞。甲状腺FNA标本研究:筛选2015年12月～2016年10月在南京军区南京总医院进行甲状腺结节细针穿刺诊断的病人标本共120例,经细胞学诊断阳性结果59例(甲状腺癌59例),阴性结果61例(良性结节)。利用细胞学试验筛选出的最佳条件观察甲状腺癌细胞和非癌细胞对IR-80染料的摄取情况,以细胞学分析为诊断金标准,计算IR-80染色方法的灵敏度和特异度,利用ROC曲线评价IR-80染色方法的诊断价值。结果:细胞系试验:当曝光时间固定时,MCF-7、VDA-VB231和MCF-10A三种细胞的NI R染色强度均随NI R染料IR-80孵育浓度的降低而逐渐降低;当IR-80孵育浓度固定时,随着曝光时间的增加,同一种细胞的NIR染料摄取增加不明显;IR-80孵育浓度和曝光时间相同时,乳腺癌细胞株MCF-7和MDA-MB231在NI R下的荧光强度高于正常乳腺上皮细胞MCF-10A。当IR-80孵育浓度为1.25μM,不同曝光条件下观察,肿瘤细胞(MCF-7和MDA-MB231)发出较强荧光,而非肿瘤细胞(MCF-10A)不被染色。筛选出IR-80应用液浓度为1.25μM,曝光时间为1s为最佳实验参数鉴别良恶性细胞。甲状腺细针穿刺(FNA)标本研究:甲状腺癌标本中的平均荧光强度明显强于对照组非甲状腺癌细胞:甲状腺癌细胞在NIR范围清楚、均匀摄取荧光;良性标本中的细胞包括良性甲状腺滤泡上皮细胞、淋巴细胞和巨噬细胞等不发出荧光。IR-80染色方法检测120例甲状腺FNA标本,有65例呈阳性结果,55例呈阴性结果,灵敏度为100%(59/59),特异度为90.1%(55/61);ROC曲线下面积为0.949,面积的标准误为0.023,IR-80染色方法结果用于判断FNA标本良恶性有显著意义(P=0.000)。结论:NIR七甲川花菁染料IR-80进行活细胞染色时,乳腺癌细胞摄取染料明显高于正常乳腺上皮细胞,IR-80染料孵育浓度为1.25μM,曝光时间为1s可用于鉴别良恶性细胞。将细胞系试验筛选出的最佳参数用于甲状腺FNA标本时,只有甲状腺癌细胞被IR-80染色,而非癌细胞不被染色,说明IR-80具有优先聚集肿瘤细胞作用,能够鉴别甲状腺FNA标本良恶性。IR-80染色方法灵敏度和特异度较高,ROC曲线表明其诊断价值高,可能成为辅助鉴别临床甲状腺FNA标本良恶性的新方法。
[Abstract]:Objective: This study first according to normal breast epithelial cell line (MCF-10A) and breast cancer cell lines (MCF-7 and VDA-MB231) on the near infrared (NIR) fluorescence intensity difference of seven methine cyanine dyes with different IR-80 intake leads to the selected dye concentration and shooting parameters to the optimal experimental conditions for differential diagnosis of benign and malignant cells; detection of IR-80 dye after incubation of thyroid carcinoma and benign thyroid nodule fluorescence intensity, compared with the traditional thyroid cytology diagnosis results, evaluation of IR-80 staining method for differential diagnosis of thyroid nodules by fine needle aspiration (FNA) specimens of benign and malignant possibility. Methods: cell lines by IR-80 dye test: application of liquid dilution with four concentration (5 M, 2.5 M, 1.25 M and 0.625 M, respectively) and human breast cancer cell lines MCF-7, MDA-MB231 and normal human breast epithelial cell line MCF-10A were incubated with fluorescence microscopy in NIR channel Under the observation of each cell (700-900nm) in 0.5s, 1s and 2S. The fluorescence intensity of exposure time to calculate the average fluorescence intensity and then use the Image J image processing software, analysis of NIR dye IR-80 on tumor cells preferentially aggregation, and selected the best experimental conditions to distinguish between benign and malignant thyroid cells. FNA study: from December 2015 to October 2016 were selected fine needle puncture in diagnosis of thyroid nodules in the General Hospital of Nanjing military region Nanjing patient samples were 120 cases, the positive cytological diagnosis results of 59 cases (59 cases of thyroid carcinoma (61 cases), negative results of benign nodules). The best conditions were screened by cytology test observation of IR-80 dye uptake in thyroid cancer and non cancer cells in cells, cytological analysis as the gold standard for diagnosis, calculate the sensitivity and specificity of IR-80 staining method, IR-80 staining method for evaluation of diagnostic value of using ROC curve values. Results: fine Cell test: when the exposure time is fixed, MCF-7 three, VDA-VB231 and MCF-10A cells NI R staining with NI R to reduce the strength of dye IR-80 incubating concentration decreased; when incubated with IR-80 concentration is fixed, with the increase of the exposure time, NIR dye uptake same cells did not increase significantly; IR-80 incubating concentration and exposure time, the fluorescence intensity of MCF-7 and MDA-MB231 in breast cancer cell line NI in R is higher than that of normal mammary epithelial cells when incubated with IR-80 MCF-10A. concentration of 1.25 M, were observed under different exposure conditions, tumor cells (MCF-7 and MDA-MB231) emit strong fluorescence, and non tumor cells (MCF-10A) don't be stained. Selected IR-80 application concentration is 1.25 M and the exposure time of 1s is the best experimental parameters in differential diagnosis of benign and malignant thyroid cells. Fine needle aspiration (FNA) specimens of thyroid carcinoma: the average fluorescence intensity was stronger than the control Group of non thyroid cancer cells: thyroid cancer cells in the range of NIR clear, uniform uptake of fluorescence; cells in benign specimens including benign thyroid follicular epithelial cells, lymphocytes and macrophages do not fluoresce.IR-80 staining method to detect 120 cases of thyroid FNA specimens, 65 cases showed positive results, 55 cases showed negative results, the sensitivity was 100% (59/59), the specificity was 90.1% (55/61); the area under the ROC curve was 0.949, the area of the standard error is 0.023. IR-80 staining results for judging FNA specimens of benign and malignant significant (P=0.000). Conclusion: NIR seven methine cyanine dyes IR-80 live cell staining, dye uptake in breast cancer cells significantly higher than that of normal mammary epithelial cells, IR-80 dye incubation concentration is 1.25 M and the exposure time of 1s can be used for differential diagnosis of benign and malignant cells. The optimal parameters of cell lines were screened for thyroid FNA specimens, only Thyroid cancer cells by IR-80 staining, and non cancer cells were not stained, indicating that IR-80 has priority to aggregation of tumor cells, can differentiate benign and malignant thyroid specimens of FNA.IR-80 staining method of sensitivity and specificity is high, ROC curve showed that the diagnostic value is high, may become auxiliary differential diagnosis of benign and malignant thyroid specimens of FNA new method.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R736.1;R581

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