微流控芯片分离胰腺癌患者循环肿瘤细胞的基础实验及其临床应用

发布时间：2018-07-01 21:49

本文选题：胰腺癌 + 循环肿瘤细胞　；参考：《中国人民解放军医学院》2016年博士论文

【摘要】：目的：1、分析上皮细胞粘附分子依赖法提取循环肿瘤细胞对其分离效率的影响;2、探索利用粒径依赖原理制备微流控芯片在胰腺癌循环肿瘤细胞分离和富集中的可行性,并建立其有效的评估体系;3、研究胰腺癌循环肿瘤细胞分离检测在其诊断和围手术期应用中的价值。材料和方法：1、收集文献,利用meta分析方法对目前胰腺癌循环肿瘤细胞相关的研究进行分析,对比上皮细胞粘附分子依赖性与非依赖性方法对其分离效率等方面的影响;2、制备微流控芯片,收集成年健康志愿者血液制备细胞系的分散系,对其进行细胞分离,利用免疫荧光染色对分离后的细胞进行计数;3、收集成年健康志愿者血液制备细胞系的分散系,分离循环肿瘤细胞,利用环介导等温扩增技术对其CK19进行检测,获取其荧光定量时间,与细胞个数进行相关性分析;4、选取胰腺良性疾病及恶性疾病的患者,对其循环肿瘤细胞CK19进行荧光定量分析,比较不同病理类型的差异性,利用操作者受试曲线评估其诊断效能;5、收集手术前后胰腺癌患者的血样标本进行循环肿瘤细胞检测和分析,评估其术前术后的差异性,以及其与手术方式、神经侵犯程度等指标的相关性。结果：1、引入研究16项,其中上皮细胞粘附分子依赖性分离方法在胰腺癌患者中的检出率为29%,非依赖法为68%,两亚组之间存在显著性差异,卡方=12.68,P=0.0004;2、成功制备微流控芯片,并成功进行Capan-1细胞系、panc-1细胞系的分选;3、利用环介导等温扩增技术对其CK19进行检测可较好评估所分选细胞的个数,与置入细胞总数之间呈显著相关;4、良性疾病与恶性疾病之间CK19荧光定量时间存在显著性差异(240.11±189.70分钟Vs 39.82±8.06分钟,t=3.165,P=0.013),在对胰腺疾病的诊断中有一定意义;5、胰腺癌手术前后其CK19荧光定量时间存在显著性差异(39.47±6.74分钟Vs 51.15±11.44分钟,t=-5.781,P0.001),无法行根治性手术的患者CK19荧光定量时间显著缩短(31±6.08分钟vs41.06±5.698分钟,t=-2.784,P=0.013)。结论：1、利用上皮细胞粘附分子依赖的方法提取循环肿瘤细胞可导致阳性率下降；2、采用粒径依赖性的微流控芯片可以实现对胰腺癌循环肿瘤细胞的无标记筛选,利用环介导等温扩增技术检测CK19的荧光定量时间,可用于评估胰腺癌患者循环肿瘤细胞分选的数目；2、胰腺良性和恶性肿瘤患者循环肿瘤细胞的CK19荧光定量时间存在显著性差异,在其病变性质的诊断中有一定价值,胰腺癌手术前后循环肿瘤细胞出现显著性差异,CK19荧光定量时间在反应患者的手术可行性具有一定价值。
[Abstract]:Objective to analyze the effect of epithelial cell adhesion molecular dependent extraction on the isolation efficiency of circulating tumor cells and to explore the feasibility of microfluidic microarray preparation based on particle size dependence in the isolation and enrichment of pancreatic cancer circulating tumor cells. An effective evaluation system was established to study the value of tumor cell separation and detection in the diagnosis and perioperative period of pancreatic cancer. Materials and methods: 1. Collect the literature and use meta analysis method to analyze the current pancreatic cancer circulating tumor cell related research. To compare the effects of epithelial cell adhesion molecular dependent and independent methods on the separation efficiency, microfluidic microarray was prepared. The blood of adult healthy volunteers was collected to prepare the cell line dispersion lines, and the cell lines were separated. The isolated cells were counted by immunofluorescence staining. The blood of adult healthy volunteers was collected to prepare the disperse cell lines. The circulating tumor cells were isolated, and their CK19 was detected by isothermal amplification technique. The fluorescence quantitative time was obtained, and the correlation between the number of cells and the number of cells was analyzed. The patients with benign and malignant diseases of pancreas were selected and the circulating tumor cells CK19 were analyzed by fluorescence quantitative analysis, and the differences of different pathological types were compared. The diagnostic efficacy was evaluated by the operator test curve. Blood samples of patients with pancreatic cancer were collected before and after operation to detect and analyze circulating tumor cells, and to evaluate the difference between them before and after operation. The correlation of nerve invasion degree and other indexes. Results among the 16 studies, the detection rate of epithelial cell adhesion molecule dependent separation method was 29% and 68%, respectively. There was significant difference between the two subgroups. The microfluidic chip was successfully prepared. And successfully carried out the separation of Capan-1 cell line panc-1 cell line. The number of the selected cells can be estimated better by the detection of CK19 by ring mediated isothermal amplification. There was a significant correlation between CK19 and the total number of implanted cells. There was a significant difference in the fluorescent time of CK19 between benign and malignant diseases (240.11 卤189.70 minutes vs 39.82 卤8.06 minutes), which had a certain significance in the diagnosis of pancreatic diseases. The fluorescence quantitative time of CK19 was significantly different (39.47 卤6.74 minutes vs 51.15 卤11.44 minutes vs 5.781g / P0.001), and that of the patients who could not undergo radical operation was significantly shortened (31 卤6.08 minutes vs41.06 卤5.698 minutes, n = 2.784 min, P 0.013), and the time of CK19 fluorescence quantification was significantly shorter in the patients who could not undergo radical surgery (39.47 卤6.74 minutes vs 51.15 卤11.44 minutes vs 5.781g / P0.001). Conclusion the positive rate of circulating tumor cells can be decreased by using the method of epithelial cell adhesion molecule dependent extraction. The particle size dependent microfluidic chip can be used to screen circulating pancreatic cancer cells without markers. The fluorescent quantitative time of CK19 was detected by using cyclized isothermal amplification technique, which can be used to evaluate the number of circulating tumor cells in patients with pancreatic cancer. 2. The fluorescent quantitative time of CK19 in circulating tumor cells was significantly different between benign and malignant tumors of pancreas, and had a certain value in the diagnosis of the nature of the disease. Significant difference of circulating tumor cells before and after pancreatic cancer operation. The fluorescent quantitative time of CK19 has some value in the feasibility of operation in patients with pancreatic cancer.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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