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Enriching and analyzing method of circulating tumor cells of colorectal cancer

A technology for tumor cells and colorectal cancer, applied in the field of medical detection and analysis, can solve the problems of inability to conduct in-depth analysis of CTC, high cost of instruments and consumables, and limitation of large-scale application, so as to improve the detection rate of CTC, low cost and low cost Effect

Inactive Publication Date: 2017-01-04
BEIJING KEXUN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the CellSearch system itself also has unsolvable defects: 1. The detection sensitivity is not high enough
Some patients with metastatic cancer cannot be captured by the CellSearch system because the CTC content in their blood is too low, or the CTC itself changes, so the detection has a high false negative rate.
2. Unable to analyze CTC in depth
In addition, the high cost of instruments and consumables also limits its further large-scale clinical application.

Method used

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  • Enriching and analyzing method of circulating tumor cells of colorectal cancer
  • Enriching and analyzing method of circulating tumor cells of colorectal cancer
  • Enriching and analyzing method of circulating tumor cells of colorectal cancer

Examples

Experimental program
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Embodiment 1

[0055] A method for enriching and analyzing colorectal cancer circulating tumor cells, comprising the following steps:

[0056] A. Collection of peripheral blood mononuclear cells

[0057] 1. Use EDTA anticoagulant tubes to collect about 10mL of patient blood samples.

[0058] 2. Using a pipette, measure 8mL of blood sample into the same labeled BD Vacutainer CPT tube.

[0059] 3. Centrifuge at 1700g room temperature for 25min.

[0060] 4. After centrifugation, the white layer under the plasma layer is monocytes and platelets. Carefully aspirate about half of the plasma, and transfer the remaining plasma and cell layer (about 2 mL) to a sterile 15 mL centrifuge tube.

[0061] 5. Add RPMI 1640 medium to the above centrifuge tube to make the total volume 10 mL. Tighten the cap of the centrifuge tube and invert the centrifuge tube 5 times to mix the cells evenly.

[0062] 6. Centrifuge at 300g for 15min and remove the supernatant.

[0063] 7. Add 1mL PBS to the above centrif...

Embodiment 2

[0130] A method for enriching and analyzing colorectal cancer circulating tumor cells, comprising the following steps:

[0131] A. Collection of peripheral blood mononuclear cells

[0132] 1. Use EDTA anticoagulant tubes to collect about 10mL of patient blood samples.

[0133] 2. Using a pipette, measure 8mL of blood sample into the same labeled BD Vacutainer CPT tube.

[0134] 3. Centrifuge at 1700g room temperature for 25min.

[0135] 4. After centrifugation, the white layer under the plasma layer is monocytes and platelets, carefully suck out about half of the plasma, and transfer the remaining plasma and cell layer (about 3mL) to a sterile 15mL centrifuge tube.

[0136] 5. Add RPMI 1640 medium to the above centrifuge tube to make the total volume 10 mL. Tighten the cap of the centrifuge tube and invert the centrifuge tube 5 times to mix the cells evenly.

[0137] 6. Centrifuge at 300g for 15min and remove the supernatant.

[0138] 7. Add 1mL PBS to the above centrifug...

Embodiment 3

[0205] A method for enriching and analyzing colorectal cancer circulating tumor cells, comprising the following steps:

[0206] A. Collection of peripheral blood mononuclear cells

[0207] 1. Use EDTA anticoagulant tubes to collect about 10mL of patient blood samples.

[0208] 2. Using a pipette, measure 8mL of blood sample into the same labeled BD Vacutainer CPT tube.

[0209] 3. Centrifuge at 1700g room temperature for 25min.

[0210] 4. After centrifugation, the white layer under the plasma layer is monocytes and platelets. Carefully suck out about half of the plasma, and transfer the remaining plasma and cell layer (about 2 mL) to a sterile 15 mL centrifuge tube.

[0211] 5. Add RPMI 1640 medium to the above centrifuge tube to make the total volume 10 mL. Tighten the cap of the centrifuge tube and invert the centrifuge tube 5 times to mix the cells evenly.

[0212] 6. Centrifuge at 300g for 15min and remove the supernatant.

[0213] 7. Add 1mL PBS to the above centrif...

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Abstract

The invention provides an enriching and analyzing method of circulating tumor cells of colorectal cancer. The enriching and analyzing method is characterized by comprising: acquiring a human body blood sample and separating single nuclear cells in the human body blood sample; carrying out two times of enriching on the single nuclear cells by adopting two types of manners of backward enriching the single nuclear cells through CD45 magnetic beads and forward enriching the single nuclear cells through EpCAM magnetic beads, so as to obtain cell suspension; after cracking the cells, extracting mRNA (messenger Ribonucleic Acid); detecting expression levels of CK20 and Survivin genes of the circulating tumor cells of the colorectal cancer through an RT-qPCR detection method; after comparing the expression levels of the two genes with an existing expression level database of the CK20 and Survivin genes of normal people and colorectal cancer patients, analyzing to know whether the circulating tumor cells of the colorectal cancer exist or not and an expression degree. The enriching and analyzing method is a method for detecting the circulating tumor cells in blood of the colorectal cancer patients and has the advantages of high sensitivity and specificity and low cost.

Description

technical field [0001] The invention belongs to the technical field of medical detection and analysis, and in particular relates to a method for enrichment and analysis of colorectal cancer circulating tumor cells. Background technique [0002] Malignant tumor metastasis is an important cause of death in cancer patients. More and more molecular biology and clinical research results show that tumor metastasis may appear in the early stage of tumorigenesis. At present, the discovery and diagnosis of tumors still rely on imaging examinations and the monitoring of traditional tumor markers clinically. It is difficult to detect tumor metastasis or recurrence early, and it is also difficult to reflect the curative effect in a timely manner. [0003] Circulating tumor cells (CTCs) are tumor cells that have shed into the blood circulation. A large number of experiments have confirmed that the detection of CTCs is helpful for monitoring tumor recurrence and metastasis, judging the pr...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12Q1/68
CPCC12N5/0693C12Q1/686C12Q1/6886C12Q2521/107C12Q2531/113
Inventor 周晔钟夏刘倩唐宇徐寒黎李伟伟李富威
Owner BEIJING KEXUN BIOTECH CO LTD
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