基于核浆比的量化成像流式技术检测肝癌病人外周血中的循环肿瘤细胞

发布时间：2018-03-22 08:34

本文选题：循环肿瘤细胞　切入点：肝癌　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：【研究目的】目前循环肿瘤细胞(circulating tumor cell,CTC)的检测方法多受限于特异性或者准确的细胞表面标记,由于这些特异的细胞表面标记只在很小的一部分细胞群体中表达,因此,通过这类方法检测到的CTC只占血液中循环的肿瘤细胞的很小一部分。在这篇文章中,我们发明了一种全新的CTC检测技术,该技术是基于检测肿瘤细胞所共有的生物特性,即细胞核浆比,而不是生物标记,如EpCAM等,因此,可以检测到更大的CTC群体,提升CTC检测的灵敏度,使检测效果具有更高的稳定性。【研究方法】首先,我们用量化成像流式Flow Sight检测肝癌患者外周血中的循环细胞,发现了一个特殊的单核细胞群体,这个细胞群体为CD45-的细胞,并且相较于其他细胞,具有更高的细胞核浆比,具有分裂能力。我们认为其为循环肿瘤细胞,并且定义为高核浆比细胞(high karyoplasmic ratios,HKR cells)。随后,我们用10例HCC伴发镜下癌栓(microvascular invasion,MVI)的病人、5例正常人的外周血进行进一步检测,以此来建立外周血HKR细胞数目与镜下癌栓出现之间的量化关系,为了增加实验的准确性,同时标记了CD45,EpCAM,DAPI标记。然后,我们随机选取了12例正常人,12例非肝癌病人(肝硬化等),52例肝癌病人(18例MVI0期的病人,18例MVI1期的病人,16例MVI2期的病人)的外周血进一步检测建立的量化关系,并以此来确立应用外周血中HKR细胞数目来预测MVI出现的模型。并且,利用受试者工作特征曲线(Reciever Operation Curves,ROC)评价了HKR细胞数目诊断MVI出现和诊断肝癌发生的灵敏度和特异度,同时,我们对入组的肿瘤病人进行了最长为期一年的术后随访,以确定我们所建立的方法在判断肿瘤预后方面的临床意义。最后,我们随机选取了42名随机病人和52名肝癌病人的外周血,来进一步检测我们所建立模型的准确性。【实验结果】在肝癌病人中,运用我们的方法检测到的外周血HKR细胞数目有56.2±23.8个/100,000个单核细胞,该值远大于非肿瘤组的病人(7.6±1.2/100,000)。在肝癌病人中,伴发MVI和不伴发MVI的病人,外周血中检测到的HKR细胞的数目也是有显著差异。通过受试者工作特征曲线分析得知,阈值是21.8/100,000,曲线下面积大于传统的检测方法(比如依赖CD45和EpCAM标记法)。这些结果表明这种新型的CTC检测技术相对于传统的方法,具有更高的敏感性和可靠性。【结论】在这项研究中,基于外周血CTCs的核浆比,我们开发了一种利用成像流式细胞仪,检测量化外周血中CTCs的方法,并对这一方法进行了验证。该技术方法依赖于检测所有肿瘤细胞所共有的细胞形态学特征-核浆比,而不是依赖特异性抗体或特异的细胞表面标志物,相比于传统的依赖细胞表面标记的CTCs检测技术,其具有更高的灵敏度,这表明它是CTC的检测和监测的一种更有效的方法。
[Abstract]:[objective] at present, the detection of circulating tumor cells is limited by specific or accurate cell surface markers, because these specific cell surface markers are expressed in a small number of cell populations. In this article, we developed a new technique for CTC detection, which is based on the biological properties common to tumor cells. That is, the ratio of nucleus to cytoplasm, rather than biomarkers, such as EpCAM, so that a larger CTC population can be detected, the sensitivity of CTC detection can be increased, and the detection effect will be more stable. [research method] first of all, We used quantitative imaging flow Flow Sight to detect circulating cells in peripheral blood of patients with liver cancer, and we found a special monocyte population of CD45- cells with a higher nuclear / cytoplasmic ratio than other cells. It is considered to be a circulating tumor cell and is defined as a high karyoplasmic cytoplasmic ratio (karyoplasmic) of HKR cells. Subsequently, we examined the peripheral blood of 5 normal controls in 10 patients with HCC accompanied by microvascular invasionus. The quantitative relationship between the number of peripheral blood HKR cells and the appearance of tumor thrombus under microscope was established. In order to increase the accuracy of the experiment, CD45 HKR was labeled with EpCAMU DAPI. We randomly selected 12 non-HCC patients (52 liver cancer patients with liver cirrhosis, 18 patients with MVI0 stage, 18 patients with MVI1 phase and 16 patients with MVI2 phase) to establish a quantitative relationship. The model of predicting the occurrence of MVI was established by using the number of HKR cells in peripheral blood. Furthermore, the sensitivity and specificity of the number of HKR cells in the diagnosis of MVI and the occurrence of HCC were evaluated by using the operating characteristic curve of the subjects. We followed up oncology patients for up to one year to determine the clinical significance of the established method in judging the prognosis of the tumor. We randomly selected the peripheral blood of 42 random patients and 52 patients with liver cancer to further test the accuracy of our model. The number of HKR cells in peripheral blood detected by our method was 56.2 卤23.8 / 100000 monocytes, which was much higher than that in non-tumor patients, 7.6 卤1.2 / 1000000.In patients with liver cancer, patients with MVI and those without MVI, The number of HKR cells detected in peripheral blood was also significantly different. The threshold is 21.8 / 100000, and the area under the curve is larger than the traditional detection methods (such as relying on CD45 and EpCAM markers). These results show that this new CTC detection technique is relative to the traditional methods. [conclusion] in this study, based on the nucleo-cytoplasmic ratio of peripheral blood CTCs, we developed a method for quantifying CTCs in peripheral blood by imaging flow cytometry. This method depends on the nuclear / cytoplasmic ratio, which is common to all tumor cells, rather than on specific antibodies or specific cell surface markers. Compared with the traditional CTCs detection technique which relies on cell surface marker, it has higher sensitivity, which indicates that it is a more effective method for detection and monitoring of CTC.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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