乳腺癌及大肠癌循环肿瘤细胞捕获及鉴定方法的建立

发布时间：2018-06-20 20:20

本文选题：乳腺癌 + 大肠癌　；参考：《浙江大学》2017年硕士论文

【摘要】：目的:利用密度梯度离心法及cytospin甩片机富集肿瘤细胞,后利用吉姆萨染色鉴定细胞形态,结合免疫荧光和免疫组化对细胞表面标志物的鉴定进一步确认是否为肿瘤细胞。从而建立较为简便的CTC捕获及鉴定方法。方法:1.肿瘤细胞的富集:在外周血中加入淋巴细胞分离液,利用密度梯度离心法进行肿瘤细胞的富集,将获得的细胞经cytospin甩片。2.肿瘤细胞的鉴定:利用吉姆萨染色确认肿瘤细胞形态,联合免疫荧光及免疫组织化学的方法利用细胞表面标志物CK及EpCAM对肿瘤细胞进行鉴定。3.对该方法的肿瘤细胞回收率进行实验,在lm丨健康人外周血中分别加入10,50,100个左右MCF-7或SW620肿瘤细胞,利用Giemsa染色方法对肿瘤细胞进行计数计算回收率。通过设立阳性对照组,阴性对照组及实验组来建立实验方法。阳性对照组为lml健康人外周血加入相应数量的肿瘤细胞,阴性对照组为1ml健康人外周血,实验组为1ml乳腺癌或结直肠癌病人外周血。从而建立具有一定的敏感性及特异性且较为简便的CTC捕获及鉴定方法。结果:阳性对照中,10/10例在Giemsa染色中可见形态较大细胞,在免疫荧光及免疫组织化学方法鉴定肿瘤细胞中可见阳性细胞。在阴性对照组中,2/10例在Giemsa染色中可见形态较大细胞,1/10例样本在免疫荧光中可见抗CK及抗EpCAM阳性细胞,但细胞形态与白细胞无明显差异,0/10例在免疫组织化学中可见抗CK及抗EpCAM阳性。有较好的敏感性及特异性。实验组中,10/10例乳腺癌及大肠癌病人外周血中用Giemsa染色可见形态较大的细胞,10/10例用免疫荧光染色可见抗CK或抗EpCAM阳性细胞,1/10例用免疫组织化学方法可见抗CK阳性细胞,细胞形态较白细胞稍大。在肿瘤细胞的回收率实验中,平均回收率为88.8%。结论:本实验对加入肿瘤细胞的健康人外周血,健康人外周血及乳腺癌或结直肠癌患者外周血进行检测,利用密度梯度离心法和cytospin富集肿瘤细胞,并联合吉姆萨染色,免疫荧光及免疫组化方法检测肿瘤细胞,表明本实验建立的方法对外周血中肿瘤细胞的探测有较好的敏感性和特异性及有较高的肿瘤细胞回收率,通过对肿瘤细胞鉴定的进一步研究具有应用于循环肿瘤细胞临床检测的价值。
[Abstract]:Aim: to enrich the tumor cells by density gradient centrifugation and cytospin flapping machine, and then to identify the cell morphology by Gimsa staining, and to identify whether the tumor cells are tumor cells by immunofluorescence and immunohistochemistry. Thus, a simple method for CTC capture and identification was established. Method 1: 1. The enrichment of tumor cells: the lymphocyte isolate was added to the peripheral blood and the tumor cells were enriched by density gradient centrifugation. The obtained cells were separated by cytospin. 2. Identification of tumor cells: the morphology of tumor cells was confirmed by Gimsa staining, and the tumor cells were identified by CK and EpCAM, combined with immunofluorescence and immunohistochemistry. The recovery rate of tumor cells was measured by Giemsa staining method. The recovery rate of tumor cells was calculated by Giemsa staining method by adding about 10,50100 MCF-7 or SW620 tumor cells into the peripheral blood of healthy individuals. The experimental method was established by setting up positive control group, negative control group and experimental group. The positive control group added corresponding tumor cells to the peripheral blood of the lml healthy people, the negative control group was the peripheral blood of the 1ml healthy people, and the experimental group was the peripheral blood of the patients with 1ml breast cancer or colorectal cancer. A simple and sensitive method for CTC capture and identification was established. Results: in 10 / 10 of the positive controls, large cells were found in Giemsa staining and positive cells in tumor cells were identified by immunofluorescence and immunohistochemistry. In the negative control group, 10 / 10 of the positive cells were found to be positive for anti-CK and anti-EpCAM in immunofluorescence in 10 / 10 of the samples with larger cells in Giemsa staining. However, there was no significant difference between cell morphology and leukocyte in 10 cases of anti-CK and anti-EpCAM positive in immunohistochemistry. It has good sensitivity and specificity. Giemsa staining was used in the peripheral blood of 10 / 10 patients with breast cancer and colorectal cancer in the experimental group. In 10 / 10 of the patients, anti-CK or anti-EpCAM positive cells could be seen in 10 / 10 of the patients with anti-CK or anti-EpCAM by immunofluorescence staining. Anti-CK positive cells could be seen in 10 / 10 of the patients with anti-CK or anti-EpCAM by immunohistochemical method. The morphology of the cells was slightly larger than that of white blood cells. The average recovery of tumor cells was 88. 8%. Conclusion: in this experiment, the peripheral blood of healthy people with tumor cells, the peripheral blood of healthy people and the patients with breast cancer or colorectal cancer were detected. The tumor cells were enriched by density gradient centrifugation and cytospin, and combined with Gimsa staining. The detection of tumor cells by immunofluorescence and immunohistochemistry showed that the method established in this study had good sensitivity and specificity for detection of tumor cells in peripheral blood and a high recovery rate of tumor cells. The further study of tumor cell identification is valuable for clinical detection of circulating tumor cells.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9;R735.34

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