一种获取循环肿瘤单细胞的方法Method for obtaining circulating tumor single cells技术领域Technical field 本发明涉及循环肿瘤细胞检测领域。 The invention relates to the field of circulating tumor cell detection.
背景技术Background technique美国肿瘤协会于2006年发表的年度报告指出，目前全世界范围内每3人中就有1人在其一生中会患有某种肿瘤，而且在发展中国家，随着污染程度的增加，患病几率还会高于1/3的比例。According to an annual report published by the American Oncology Association in 2006, one in three people worldwide currently has a certain type of tumor in their lifetime, and in developing countries, as the degree of pollution increases, The chance of illness will be higher than 1/3proportion.
目前全世界用于肿瘤诊断的方法可归为三大类：病理学（活检切片）；影像学（超声、X光、CT或PET等）和血清学（血清肿瘤相关蛋白，如CA-125、CA-199或CEA等），但所有这些方法都有不可避免的缺点，例如灵敏性及特异性差和过于依赖医生的主观判断等。因此目前临床急需一种能够为医生及病人提供准确，快速的肿瘤检测手段。经过长时间的大量研究，美国FDA认证的人外周血循环肿瘤细胞（CirculatingTumorCell，简称CTC）检测目前已被公认为最好及最客观的检测手段之一。目前已有越来越多的国内外医生开始应用此技术作为肿瘤检查的重要辅助手段。不仅于此，CTC检测也被公认为是目前国外新近流行的肿瘤治疗方法—即阻断肿瘤细胞入血转移的唯一有效检测指标。除了上述CTC可作为肿瘤检测手段外，CTC还被证明可成功应用于以下一系列其它方面的用途：(1)体内化疗药物药效的快速评估: 相对于每12周一查的CT诊断，CTC在第2周即可显示药效结果，这一点对临床医生及挽救病人生命尤为重要；(2)根据CTC数目，判断病人愈后状况及存活时间：美国Immunicon/Veridex (Johnson & Johnson)公司经过在数百肿瘤病人标本上的严格验证，已可根据治疗前后的CTC数目变化准确地预测乳腺癌、结直肠癌及前列腺癌病人的存活时间，这一点已被美国FDA认证接受，开始在美国众多医院中被广泛应用；(3)肿瘤病人复发的监测与及时诊断：绝大多数的肿瘤复发从根本上讲实际就是肿瘤转移过程，因而通过对治疗后的病人CTC的持续检测，可以达到监测病人肿瘤复发的目的；（4）个体化治疗的体外肿瘤治疗药物筛选；及（5）正常人群体检普查以利于尽可能的肿瘤早期诊断等。除了应用于临床外，CTC亦可应用于众多药厂和科研机构的基础研究，包括寻找新的肿瘤标记物以及开发新的抗肿瘤药物等。At present, the methods used for tumor diagnosis in the world can be classified into three categories: pathology (biopsy); imaging (ultrasound, X-ray, CT)Or PET, etc.) and serology (serum tumor-associated proteins, such as CA-125,CA-199 or CEA, etc., but all of these methods have inevitable shortcomings, such as poor sensitivity and specificity and too much dependence on the subjective judgment of the doctor. Therefore, there is an urgent need for an accurate and rapid tumor detection method for doctors and patients. After a long period of extensive research, FDA-approved human peripheral blood circulating tumor cells (Circulating)TumorCell, referred to as CTC), is currently recognized as one of the best and most objective detection methods. At present, more and more doctors at home and abroad have begun to apply this technology as an important auxiliary means for tumor examination. Not only that, CTC testing is also recognized as the only popular cancer treatment method in foreign countries - the only effective detection index to block tumor cells into blood transfer. In addition to the above CTC as a means of tumor detection, CTC has also been proven to be successfully applied to a range of other applications: (1)Rapid assessment of the efficacy of in vivo chemotherapy drugs: Compared to CT diagnosis every 12 weeks, CTC can show efficacy results in the second week, which is especially important for clinicians and saving patients' lives; (2)According to the number of CTCs, judge the patient's recovery status and survival time: US Immunicon/Veridex (Johnson & Johnson)After rigorous verification on hundreds of tumor patient specimens, the company has been able to accurately predict the survival time of breast cancer, colorectal cancer and prostate cancer patients based on the number of CTCs before and after treatment. This has been accepted by the US FDA and has begun to It is widely used in many hospitals in the United States; (3) Surveillance and timely diagnosis of recurrence of tumor patients: The vast majority of tumor recurrences are fundamentally the process of tumor metastasis, and thus can be achieved by continuous detection of CTCs in patients after treatment. The purpose of monitoring tumor recurrence of patients; (4) screening of in vitro tumor treatment drugs for individualized treatment; and (5) screening of normal populations to facilitate early diagnosis of tumors. In addition to its clinical application, CTC can also be used in basic research in a variety of pharmaceutical and scientific research institutions, including the search for new tumor markers and the development of new anti-tumor drugs.
目前美国FDA已于2004年认证了美国上市公司Immunicon/Veridex的CTC检测技术及其临床应用，该技术目前已应用于美国多家医院的循环肿瘤细胞检测。At present, the US FDA has certified the CTC detection technology and clinical application of the US listed company Immunicon/Veridex in 2004. The technology has been applied to the detection of circulating tumor cells in many hospitals in the United States.
但是，Immunicon/Veridex的检测技术存在显而易见的缺陷。该技术分离检测肿瘤细胞主要是基于肿瘤细胞表面的一种特定标志物。但众所周知，此肿瘤细胞表面标志物随着肿瘤细胞病理性生长及肿瘤细胞的入血而异常表达。而且，此特定标志物在细胞表面的表达量也因肿瘤不同而差异极大，包括高表达与不表达。所有这些使得该技术在很多情况下捕获不到肿瘤细胞，从而限制了该技术的广泛普及与应用。However, Immunicon/Veridex's detection technology has obvious drawbacks. This technique separates and detects tumor cells based primarily on a specific marker on the surface of tumor cells. However, it is well known that this tumor cell surface marker is abnormally expressed along with the pathological growth of tumor cells and the entry of blood into tumor cells. Moreover, the expression level of this specific marker on the cell surface also varies greatly depending on the tumor, including high expression and no expression. All of this makes the technology incapable of capturing tumor cells in many cases, thus limiting the widespread popularity and application of this technology.
发明内容Summary of the invention本发明提供一种便于进行循环肿瘤细胞检测和研究的获取循环肿瘤单细胞的方法。The present invention provides a method for obtaining circulating tumor cells by facilitating detection and research of circulating tumor cells.
本发明提供一种获取循环肿瘤单细胞的方法，包括如下步骤：The invention provides a method for obtaining circulating tumor single cells, comprising the following steps:
a) 富集体液样本中的循环肿瘤细胞；a) circulating tumor cells in a sample of concentrated fluid;
b) 识别出所述循环肿瘤细胞；b) identifying the circulating tumor cells;
c) 分离出单个循环肿瘤细胞。c) A single circulating tumor cell is isolated.
通过去除体液样本中的非循环肿瘤细胞物质，来提高循环肿瘤细胞Improve circulating tumor cells by removing non-circulating tumor cell material from body fluid samples
的浓度，起到富集循环肿瘤细胞的作用。在富集后的体液样本中，识别筛选出循环肿瘤细胞；将识别筛选出的循环肿瘤细胞一个个分离出来，便于后续的检测和研究。The concentration plays a role in enriching circulating tumor cells. In the enriched body fluid samples, the circulating tumor cells are identified and screened; the identified circulating tumor cells are separated one by one, which facilitates subsequent detection and research.
体液一般指细胞外液。Body fluid generally refers to extracellular fluid.
进一步的，所述步骤a)中，通过去除所述体液样本中的白细胞和红Further, in the step a), by removing white blood cells and red in the body fluid sample
细胞来富集所述循环肿瘤细胞。The cells are enriched for the circulating tumor cells.
进一步的，所述步骤a)中，通过离心技术去除所述体液样本中的红Further, in the step a), the red in the body fluid sample is removed by centrifugation
细胞，通过抗体磁珠标记技术去除所述体液样本中的白细胞。Cells, which remove leukocytes from the body fluid sample by antibody magnetic bead labeling techniques.
进一步的，所述步骤b)中，利用免疫荧光染色法，用上皮细胞特异性表面标志识别出所述循环肿瘤细胞。Further, in the step b), the circulating tumor cells are identified by an epithelial cell-specific surface marker by immunofluorescence staining.
进一步的，所述步骤b)中，利用微流控芯片，通过比较肿瘤细胞和正常细胞的粒径大小来识别出所述循环肿瘤细胞。Further, in the step b), the circulating tumor cells are identified by comparing the particle size of the tumor cells and the normal cells by using a microfluidic chip.
进一步的，所述步骤b)中，利用肿瘤细胞表面标志和荧光原位杂交法识别出所述循环肿瘤细胞。Further, in the step b), the circulating tumor cells are identified by a tumor cell surface marker and a fluorescence in situ hybridization method.
进一步的，所述循环肿瘤细胞是泌尿系统肿瘤细胞，所述肿瘤细胞表面标志是膀胱癌的OCT基因、膀胱癌的FGFR3基因、膀胱癌的Ras基因或肾癌的VHL基因；识别时，利用两个或两个以上的特异性表面标志进行识别鉴定。Further, the circulating tumor cell is a urinary system tumor cell, and the tumor cell surface marker is an OCT gene of bladder cancer, a FGFR3 gene of bladder cancer, a Ras gene of bladder cancer or a VHL gene of kidney cancer; Identification of one or more specific surface markers.
进一步的，所述步骤c)中，利用微吸管分离出单个循环肿瘤细胞。Further, in the step c), a single circulating tumor cell is isolated by using a micropipette.
本发明的有益效果是：通过将体液样本中的循环肿瘤细胞一个个单独分离出来，从而便于后续的检测和科学研究。The beneficial effects of the present invention are: by separately separating the circulating tumor cells in the body fluid sample, thereby facilitating subsequent detection and scientific research.
具体实施方式detailed description下面通过具体实施方式对本发明作进一步详细说明。The invention will now be further described in detail by way of specific embodiments.
一种获取循环肿瘤单细胞的方法，首先，利用差减法在体液样本中A method for obtaining single cells of circulating tumors, firstly, using a subtractive method in a body fluid sample
富集循环肿瘤细胞(CTC)；接着，识别出循环肿瘤细胞；最后，分离出单个循环肿瘤细胞。The circulating tumor cells (CTC) are enriched; next, circulating tumor cells are identified; finally, a single circulating tumor cell is isolated.
在一种实施方式中，该方法包括如下步骤：a)利用现有离心技术去掉肿瘤患者体液样本中的红细胞，利用现有抗体磁珠标记技术移除白细胞，从而在该体液样本中富集循环肿瘤细胞；b)通过免疫荧光染色法，用上皮细胞特异性标志识别出循环肿瘤细胞；c)采用微吸管，在荧光显微镜下操作，吸出单个循环肿瘤细胞。分离出的单个循环肿瘤细胞即可用于肿瘤分析检测或其他科学研究。In one embodiment, the method comprises the steps of: a) removing red blood cells from a body fluid sample of a tumor patient using existing centrifugation techniques, and removing white blood cells using existing antibody magnetic bead labeling techniques to enrich the circulation in the body fluid sample Tumor cells; b) Identifying circulating tumor cells by immunofluorescence staining with epithelial cell-specific markers; c) aspirating a single circulating tumor cell using a micropipette under a fluorescence microscope. The isolated single circulating tumor cells can be used for tumor analysis or other scientific research.
本实施方式中，抗体磁珠标记技术即利用荧光标记抗体包被的磁珠与白细胞偶联，来去除体液样本中的白细胞。由于白细胞含有单核细胞、粒细胞、巨噬细胞和淋巴细胞等，可以利用不同的抗体组合来去除白细胞。In the present embodiment, the antibody magnetic bead labeling technique is to couple the magnetic beads coated with the fluorescently labeled antibody to the white blood cells to remove the white blood cells in the body fluid sample. Since leukocytes contain monocytes, granulocytes, macrophages, and lymphocytes, different combinations of antibodies can be used to remove white blood cells.
本实施方式中，微吸管的直径一般为微米级。吸出操作时，可以采用人工用嘴轻吸的方式。由于采用微吸管，可以有效的控制吸力，使吸力不会过大。In the present embodiment, the diameter of the micropipette is generally on the order of micrometers. When sucking out, it can be manually sucked by the mouth. Thanks to the use of a micropipette, the suction can be effectively controlled so that the suction is not excessive.
在另一种实施方式中，该方法包括如下步骤：a)利用离心技术去除肿瘤患者体液样本中的红细胞，利用抗体磁珠标记技术去除体液样本中的白细胞，从而在该体液样本中富集循环肿瘤细胞；b)采用微流控芯片，通过比较肿瘤细胞和正常细胞的粒径大小来识别出循环肿瘤细胞；c)分离出单个循环肿瘤细胞。In another embodiment, the method comprises the steps of: a) removing red blood cells from a body fluid sample of a tumor patient by centrifugation, and removing leukocytes in the body fluid sample by using an antibody magnetic bead labeling technique, thereby enriching the circulation in the body fluid sample; Tumor cells; b) using a microfluidic chip to identify circulating tumor cells by comparing the particle size of tumor cells and normal cells; c) isolating a single circulating tumor cell.
本实施方式中，正常细胞具有粒径范围，通过比较肿瘤细胞的粒径和该粒径范围，如该粒径在该粒径范围之外，则是肿瘤细胞。In the present embodiment, the normal cells have a particle size range, and by comparing the particle diameter of the tumor cells with the particle size range, if the particle diameter is outside the particle size range, it is a tumor cell.
在又一种实施方式中，该方法包括如下步骤：a) 在体液样本中富集循环肿瘤细胞；b)利用现有肿瘤细胞表面标志和荧光原位杂交法识别出泌尿系统肿瘤细胞；c) 采用微吸管，在荧光显微镜下操作，吸出单个循环肿瘤细胞。分离出的单个循环肿瘤细胞即可用于肿瘤分析检测或其他科学研究。In yet another embodiment, the method includes the steps of: a)Enrichment of circulating tumor cells in body fluid samples; b) identification of urinary tumor cells using existing tumor cell surface markers and fluorescence in situ hybridization; c)Single circulating tumor cells were aspirated using a micropipette operating under a fluorescence microscope. The isolated single circulating tumor cells can be used for tumor analysis or other scientific research.
本实施方式中，识别泌尿系统肿瘤细胞时，泌尿系统肿瘤细胞表面标志如膀胱癌的OCT、FGFR3或Ras基因、如肾癌的VHL基因等。具体识别时，可以使用膀胱癌特异性表面标志识别膀胱癌细胞，如以OCT4、FGFR3或Ras等多基因作为多个靶点进行识别，即能够利用两个或两个以上的特异性表面标志进行识别鉴定。In the present embodiment, when the urinary system tumor cells are recognized, the urinary system tumor cell surface markers such as OCT, FGFR3 or Ras genes of bladder cancer, such as the VHL gene of renal cancer, and the like. In specific recognition, bladder cancer-specific surface markers can be used to identify bladder cancer cells, such as multi-genes such as OCT4, FGFR3 or Ras as multiple targets for identification, ie, two or more specific surface markers can be used. Identification and identification.
以上内容是结合具体的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换。The above is a further detailed description of the present invention in connection with the specific embodiments, and the specific embodiments of the present invention are not limited to the description. A number of simple derivations or substitutions may be made by those skilled in the art without departing from the inventive concept.