循环肿瘤细胞分离方法的建立和临床应用的探索

发布时间：2018-10-18 17:18

【摘要】：目的采用Opti Prep密度梯度离心结合免疫磁珠负向筛选法富集循环肿瘤细胞(Circulating Tumor Cell,CTC),并联合多色免疫荧光标记和荧光原位杂交技术检测CTC,建立一种简易高效的富集和鉴定癌症患者外周血CTC的方法,有效应用于临床肿瘤患者外周血的CTC计数检测。在此基础上,进一步探索建立单细胞捕获技术平台,对特定CTC细胞或者CTC细胞群进行单细胞组学的研究。显微镜观察到CTC后进行显微切割和显微吸取以获取单细胞进行分子鉴定,这一研究将促进CTC的分子鉴定,开发新的治疗靶点,并产生对癌症患者更有效的治疗方案。方法1.利用不同密度细胞的沉降系数不同,配比不同浓度的Opti Prep溶液梯度离心分离外周血各细胞成分,验证单核细胞聚集细胞层。分别选取QGY-7701等多种肿瘤细胞混入外周血,模拟体内循环肿瘤细胞状态。比较Opti Prep液与Ficoll液富集肿瘤细胞的效率、回收率、活性率。密度梯度离心结合免疫磁珠建立最优的循环肿瘤细胞富集体系。2.选取5种肿瘤细胞株:肝癌细胞(QGY-7701、Huh-7、SMMC-7721)、卵巢癌细胞HO-8910、肺癌细胞A549分别进行CD45和CK18两种抗体的细胞免疫荧光染色,CIK细胞作对照。FISH标记CIK细胞染色体数目。将肿瘤细胞混入正常人外周血模拟外周血循环肿瘤细胞进行富集,免疫荧光技术结合FISH检测,准确识别肿瘤细胞。3.联合富集和检测方法精确检测癌症患者的外周血循环肿瘤细胞,进行方法学验证评估。并根据患者CTC检测数目和临床预后数据分析我们建立的CTC检测对于肿瘤患者预后判断的作用。4.尝试显微切割和显微吸取提取单个靶细胞及较纯的目的细胞群,应用Carl Zeiss PALM Combi System和Cell Ector Plus两大系统捕获单个细胞,并进行RT-PCR检测肿瘤细胞的基因表达情况。结果1.Opti Prep液能有效分离单核细胞,将QGY-7701肿瘤细胞富集于白膜层。肝癌细胞(QGY-7701、Huh-7、SMMC-7721)、卵巢癌细胞HO-8910、肺癌细胞A549在Opti Prep中肿瘤细胞的富集效率均高于Ficoll,两组间差异均有统计学意义(P0.05)。单细胞操作取50个左右肿瘤细胞入血模拟循环肿瘤细胞,比较Opti Prep与Ficoll的回收率较一致。2.免疫荧光结果显示,仅CIK细胞观察到CD45红色荧光,而CK18则是仅CIK细胞无绿色荧光。荧光原位杂交结果表明能显著标记细胞染色体,以此识别染色体异常细胞。3.通过对33例入组患者外周血的检验验证以上富集和检测循环肿瘤细胞的方法,均发现有循环肿瘤细胞的存在,说明建立的这一方法能有效检出癌症患者血液中的CTC。实验结果显示,CTC数量较多对应较短生存期,较差预后。4.本研究根据实验需要,涉及两种可识别荧光标记的单细胞获取技术,结果显示均能迅速准确的得到单个目的细胞,解决细胞异质性问题,获取纯净靶细胞,进而为少量CTC的下一步分子鉴定提供了机会,显示了良好的应用前景。结论本研究成功建立了Opti Prep密度梯度离心结合免疫磁珠负向筛选法富集CTC,并联合多色免疫荧光标记结合荧光原位杂交技术检测CTC,有效应用于临床肿瘤患者外周血的CTC鉴定。另外,尝试单细胞捕获新技术获取稀有循环肿瘤细胞,为后续单细胞目的基因表达鉴定和进一步的基因测序研究探寻方向,为癌症的预后及预测信息指明方向、为个体化指导治疗和用药的合理选择提供参考。
[Abstract]:Objective To enrich circulating tumor cells (CTC) with Opti Prep density gradient centrifugation combined with immunomagnetic beads, and to establish a simple and efficient method for enriching and identifying peripheral blood CTC in cancer patients by combining multi-color immunofluorescence labeling and fluorescence in situ hybridization technique. The effect was used in the detection of CTC counts in peripheral blood of patients with clinical tumors. On the basis of this, a single cell acquisition technology platform was established to study the single cell group study of specific CTC cells or CTC cells. Microdissection and microscopic aspiration after the CTC were observed to obtain single cells for molecular identification, which would facilitate molecular identification of CTCs, develop new therapeutic targets, and produce a more effective treatment regimen for cancer patients. Method 1. The cell components of peripheral blood were obtained by gradient centrifugation of Opti Prep solution with different densities and different density cells. Multiple tumor cells, such as QGY-7701, were selected to be mixed with peripheral blood to simulate the state of circulating tumor cells in vivo. The efficiency, recovery and activity of Opti Prep liquid and Ficoll liquid enriched tumor cells were compared. The optimal circulating tumor cell enrichment system was established by density gradient centrifugation combined with immunomagnetic beads. Five tumor cell lines were selected: liver cancer cell (QGY-7701, Huh-7, NCI-7721), ovarian cancer cell HO-8910, lung cancer cell A549, and cell immunofluorescent staining of CD45 and CK18 antibodies respectively. CIK cells were used as control. FISH markers CIK cell chromosome number. the tumor cells are mixed with normal human peripheral blood to simulate peripheral blood circulating tumor cells to enrich, the immunofluorescence technique is combined with FISH detection, and the tumor cells are accurately identified. Combined enrichment and detection methods accurately detect peripheral blood circulating tumor cells in cancer patients and conduct methodological verification evaluation. Based on the patient's CTC test number and clinical outcome data, we established the effect of CTC detection on the prognosis of patients with tumor. Single target cell and pure target cell population were extracted by microdissection and microdissection. The single cell was captured by Carl Zeiss PALM Combi System and Cell Ector Plus, and the gene expression of tumor cells was detected by RT-PCR. Results 1. Opti Prep liquid could effectively separate monocytes and enrich QGY-7701 tumor cells in white membrane. The concentration of tumor cells in Oti Prep was higher than that of Ficoll (P0.05). In single cell operation, 50 or so tumor cells were injected into the blood-simulated circulating tumor cells, and the recovery rates of Opti Prep and Ficoll were more consistent. Immunofluorescence showed that only CIK cells observed CD45 red fluorescence, while CK18 was only CIK cells without green fluorescence. The results of fluorescence in situ hybridization showed that the cell chromosome could be marked by fluorescence in situ hybridization. By examining the methods of enriching and detecting circulating tumor cells in the peripheral blood of 33 patients enrolled, the existence of circulating tumor cells was found, which indicated that the established method could effectively detect the CTC in the blood of cancer patients. The results showed that the number of CTC corresponded to shorter survival and poor prognosis. According to the experimental requirements, the study relates to two single cell acquisition technologies capable of identifying fluorescent labels, and the results show that the single cell can be rapidly and accurately obtained, the problem of cell heterogeneity is solved, pure target cells are obtained, and the next step molecular identification of a small amount of CTCs is provided with the opportunity, and a good application prospect is displayed. Conclusion This study successfully established Opti Prep density gradient centrifugation combined with immunomagnetic beads to enrich CTC, and combined with fluorescent in situ hybridization technique to detect CTC, and has the effect of CTC identification in peripheral blood of clinical tumor patients. in addition, attempt to acquire rare circulating tumor cells by single cell capture novel technology, identify and further gene sequencing research search directions for subsequent single cell target gene expression identification and further gene sequencing, indicate that prognosis and prediction information of cancer refer to a bright direction, Provide reference for the reasonable choice of individualized instruction therapy and medication.
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R730

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