新型循环肿瘤细胞检测方法在头颈部恶性肿瘤中的临床应用
发布时间：2018-09-17 18:29
【摘要】：目的循环血液中活性肿瘤细胞的检测被看作是液体活检,具有动态监测的功能,在肿瘤早期诊断、个体化治疗、疗效评价、预后判断等方面具有重要的临床意义,然而由于其在血液中数量很少,循环肿瘤细胞(circlulating tumor cells,CTCs)的分离和鉴定成为临床应用的难点。复制选择性溶肿瘤腺病毒Ad11-5ETel-GFP是一种通过同源重组获得的病毒,可以选择性的在具有端粒酶活性的细胞内复制并表达绿色荧光蛋白(Green Fluorescent Protein,GFP)。本研究旨在利用该病毒捕获循环肿瘤细胞,从而建立一种可重复、简单、无创的检测方法,并对该检测方法的可靠性和临床应用价值进行探讨。方法首先通过实验验证我们所建立的方法能够有效捕获到肿瘤细胞,包括:将AD11-5ETel-GFP病毒加入正常人外周血中,观察GFP在正常白细胞的表达情况;将A549(肺癌细胞系),SKBR3(乳腺癌细胞系)分别以10、50、100个细胞三个梯度加入1ml人外周血中,建立循环肿瘤细胞的模型并检测其回收率;使用抗人CD45抗体进行细胞免疫染色,荧光显微镜下观察结果。以上结果说明本实验建立的新型检测方法能有效的捕获循环细胞。将该方法应用于头颈部恶性肿瘤的患者,收集临床标本48例,将标本中红细胞裂解,加入病毒于37℃条件下孵育24h,固定涂片后于荧光显微镜下观察计数。并分析不同临床参数头颈部肿瘤患者术前外周血CTCs的检出情况。结果1.复制选择性溶肿瘤病毒不感染正常人白细胞。2.分别于正常人外周血中加入的10,50,100个A549、SKBR3细胞,其检出率分别为77.5%,84.0%,80.25%;80.0%,82.5%,81.25%,应用pearson卡方检验比较两组间差异,结果显示用溶肿瘤腺病毒法检测两组不同的肿瘤细胞,其检出率无显著差异,即本实验所研究的方法对各肿瘤细胞系均敏感且回收率较高。并通过细胞免疫染色实验进一步验证所捕获的细胞为循环肿瘤细胞。3.在收集的48例头颈部恶性肿瘤的临床标本中,19例患者CTCs阳性,检出率为39.6%,且CTCs的检出率与性别、年龄、肿瘤分期、肿瘤的发病部位、淋巴结转移与否等临床参数无关,与肿瘤分化程度相关,肿瘤分化越差,CTCs的检出率越高。结论本研究建立了一种新型循环肿瘤细胞的检测方法,初步确定了溶肿瘤腺病毒检测CTCs的标准,并对该方法的可靠性及临床应用价值进行了评估。该方法能够特异性示踪循环肿瘤细胞,具有重要的临床应用价值。
[Abstract]:Objective the detection of active tumor cells in circulating blood is regarded as fluid biopsy, which has the function of dynamic monitoring. It has important clinical significance in early diagnosis of tumor, individualized treatment, evaluation of curative effect and judgment of prognosis. However, the isolation and identification of circulating tumor cells (circlulating tumor cells,CTCs) has become a difficulty in clinical application due to its small number in the blood. Replication-selective adenovirus (Ad11-5ETel-GFP) is a homologous recombinant virus that can selectively replicate and express green fluorescent protein (Green Fluorescent Protein,GFP) in cells with telomerase activity. The purpose of this study was to establish a repeatable, simple and noninvasive detection method by using the virus to capture circulating tumor cells, and to discuss the reliability and clinical application value of the detection method. Methods the method was proved to be effective in capturing tumor cells, including: AD11-5ETel-GFP virus was added into the peripheral blood of normal people to observe the expression of GFP in normal white blood cells; A549 (lung cancer cell line) SKBR3 (breast cancer cell line) was added to the peripheral blood of 1ml with three gradients of 10,50100 cells, and the model of circulating tumor cells was established and the recovery rate was detected. The results were observed under fluorescence microscope. The above results show that the new detection method established in this experiment can capture circulating cells effectively. The method was applied to the patients with head and neck malignant tumor. 48 clinical specimens were collected. The red blood cells were lysed in the specimens and incubated with the virus at 37 鈩，
本文编号：2246752
[Abstract]:Objective the detection of active tumor cells in circulating blood is regarded as fluid biopsy, which has the function of dynamic monitoring. It has important clinical significance in early diagnosis of tumor, individualized treatment, evaluation of curative effect and judgment of prognosis. However, the isolation and identification of circulating tumor cells (circlulating tumor cells,CTCs) has become a difficulty in clinical application due to its small number in the blood. Replication-selective adenovirus (Ad11-5ETel-GFP) is a homologous recombinant virus that can selectively replicate and express green fluorescent protein (Green Fluorescent Protein,GFP) in cells with telomerase activity. The purpose of this study was to establish a repeatable, simple and noninvasive detection method by using the virus to capture circulating tumor cells, and to discuss the reliability and clinical application value of the detection method. Methods the method was proved to be effective in capturing tumor cells, including: AD11-5ETel-GFP virus was added into the peripheral blood of normal people to observe the expression of GFP in normal white blood cells; A549 (lung cancer cell line) SKBR3 (breast cancer cell line) was added to the peripheral blood of 1ml with three gradients of 10,50100 cells, and the model of circulating tumor cells was established and the recovery rate was detected. The results were observed under fluorescence microscope. The above results show that the new detection method established in this experiment can capture circulating cells effectively. The method was applied to the patients with head and neck malignant tumor. 48 clinical specimens were collected. The red blood cells were lysed in the specimens and incubated with the virus at 37 鈩，
本文编号：2246752
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