A negative enrichment method for circulating tumor cells

A negative enrichment and tumor cell technology, applied in the field of molecular biology, can solve the problems of low recovery rate of circulating tumor cells, difficulty in selection of biomarkers, inability to obtain active ones, etc., to achieve shortened enrichment time and high sensitivity , the effect of reducing dosage

Active Publication Date: 2020-06-02
亚能生物技术(深圳)有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0018] The technical problem to be solved by the present invention is: to provide a negative enrichment method for circulating tumor cells, which solves the problem of low recovery rate of circulating tumor cells and inability to obtain active circulating tumor cells and biomarkers in existing circulating tumor cell separation methods. Problems with difficult selection and complex separation techniques

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  • A negative enrichment method for circulating tumor cells
  • A negative enrichment method for circulating tumor cells
  • A negative enrichment method for circulating tumor cells

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Embodiment 1

[0062] A circulating tumor cell negative enrichment method, comprising the following steps:

[0063] S1: Add density gradient separation medium to the lymphocyte separation tube, and centrifuge instantaneously so that the density gradient separation medium is completely centrifuged to the bottom of the tube; add 3 mL of blood samples from tumor patients to the lymphocyte separation tube filled with density gradient separation 3mL of washing buffer, for the first centrifugation, the condition of the first centrifugation is 25°C, 800g / 15min;

[0064] S2: Transfer the upper layer solution in the baffle plate of the above-mentioned lymphocyte separation tube after the first centrifugation to the first centrifuge tube, and perform the second centrifugation. The conditions of the second centrifugation are 25°C, 300g / 10min, suction Remove the supernatant, add 5mL of erythrocyte lysate, let stand for 5min;

[0065] S3: Centrifuge the first centrifuge tube after standing still for the...

Embodiment 2

[0070] Please refer to figure 1 , mixed a certain number of SiHa cells in the peripheral blood of healthy people to simulate the blood samples of patients with cervical cancer containing circulating tumor cells, and evaluated the enrichment efficiency of circulating tumor cell negative enrichment method established, the specific monitoring steps are as follows :

[0071] 1. Pre-label the cultured and digested SiHa cells with Celltracker Green for green fluorescence, and dilute in proportion to obtain 2×10 4 The cell suspension was accurately counted under a fluorescent microscope and adjusted to 100 cells / 5uL;

[0072] 2. Mix the counted cell suspension into 3 mL of peripheral blood to prepare peripheral blood samples containing 100 SiHa cells / 3 mL;

[0073] 3. For peripheral blood samples containing 100 SiHa cells / 3mL, carry out negative enrichment of circulating tumor cells according to the following two schemes respectively;

[0074] A. Traditional density gradient centr...

Embodiment 3

[0078] Please refer to figure 2 and image 3 A certain amount of ovarian cancer SKOV-3, cervical cancer SiHa cells, and breast cancer SKBR3 cells were mixed into the peripheral blood of healthy people to evaluate whether the negative enrichment method of circulating tumor cells involved in this protocol is applicable to circulating tumor cells of different tumor types Detection, the specific detection steps are as follows:

[0079] 1. In this example, 100 ovarian cancer SKOV-3, cervical cancer SiHa cells, and breast cancer SKBR3 cells were mixed into the peripheral blood of healthy people to simulate blood samples containing circulating tumor cells from different tumor patients, cultured, digested and harvested Cells of different tumor types were pre-labeled with Celltracker Green, diluted in proportion to obtain cell suspensions of different concentrations, and counted accurately under a fluorescent microscope.

[0080] 2. The counted cell suspension was mixed into 3 mL of...

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Abstract

The invention relates to the field of molecular biology, and in particular relates to a circulating tumor cell negative enrichment method. The method comprises the following steps that pretreatment is carried out on a blood sample by adopting density gradient centrifugation on the basis of a lymphocyte separating tube, so that the operating step of separating leukocytes is simplified, the enrichment time of the circulating tumor cell is shortened, and the whole operation is competed within 2.5h; the number of the left leukocytes is 30-50 % of that of original leukocytes of the blood sample, self-made negative enrichment immunomagnetic beads CD45 are adopted for marking the leukocytes and removed, so that the used number of the immunomagnetic beads is reduced, and the cost for negative enrichment of the circulating tumor cells is greatly saved. The lymphocyte separating tube is adopted for carrying out pretreatment on the blood sample, the negative enrichment method of the circulating tumor cell is improved, the stability of the enrichment operation is effectively improved, and the recovery ratio of the circulating tumor cell achieves 85% or above. The circulating tumor cell negative enrichment method further has the advantage of high sensitivity, and the circulating tumor cells can be stably detected within a few samples.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a negative enrichment method for circulating tumor cells. Background technique [0002] Circulating tumor cells (CTCs) refer to tumor cells released from solid tumors or metastases into the peripheral blood circulation spontaneously or due to diagnostic and therapeutic procedures, and are the main cause of distant metastasis in patients with malignant tumors. According to statistics, metastasis is the cause of death of more than 90% of tumor patients. In recent years, with the continuous improvement of related technologies in some basic disciplines, CTC detection, as a new type of non-invasive diagnostic tool, has the advantages of non-invasiveness, multiple acquisitions, and real-time monitoring compared with traditional tissue biopsy. Vividly called "liquid biopsy", it has important clinical research and application value, and has become a hot spot in clinical research. A lar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2501/599C12N2509/00
Inventor 任晓辉黄慧玲杨昂刘晶晶李印淑张磊
Owner 亚能生物技术(深圳)有限公司
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