CN111855541A - Circulating tumor cell detection reagent, kit and detection method - Google Patents
Circulating tumor cell detection reagent, kit and detection method Download PDFInfo
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- CN111855541A CN111855541A CN201910343600.0A CN201910343600A CN111855541A CN 111855541 A CN111855541 A CN 111855541A CN 201910343600 A CN201910343600 A CN 201910343600A CN 111855541 A CN111855541 A CN 111855541A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/34—Purifying; Cleaning
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N2015/1006—Investigating individual particles for cytology
Abstract
The invention relates to a circulating tumor cell detection reagent, a kit and a detection method, because 5-ALA and enriched tumor circulating cells can generate a specific metabolite protoporphyrin PpIX in the in-vitro culture process, the protoporphyrin can generate red fluorescence at the wavelength of 635nm, and the circulating tumor cells can be counted by a fluorescence microscope by utilizing the characteristic, so that the circulating tumor cells in peripheral blood can be accurately detected.
Description
Technical Field
The invention relates to a cell detection reagent and a cell detection method, in particular to a circulating tumor cell detection reagent, a circulating tumor cell detection kit and a circulating tumor cell detection method.
Background
After a tumor occurs in a human body, a part of tumor cells enter the peripheral blood circulation of the human body due to the growth and apoptosis of the tumor cells, and the part of tumor cells are called Circulating Tumor Cells (CTCs). Detection of Circulating Tumor Cells (CTCs) facilitates early diagnosis of tumors and may also be used in conjunction with diagnosis following treatment to assess efficacy.
The existing circulating tumor cell detection methods are various, generally, peripheral blood is collected to enrich tumor cells, and then qualitative and quantitative detection is carried out through morphology or gene sequencing, so that the method is complicated, and the accuracy and timeliness are still to be improved.
5-aminolevulinic acid (5-aminolevulinic acid), formula: c5H9N03Molecular weight 131.13, CAS number: 106-60-5, 5-ALA is a prefix compound of tetrahydropyrrole (tetrahydropyrrole is a substance forming heme, cytochrome, vitamin B12), and is used for synthesizing chlorophyll, heme, vitamin A, vitamin B, vitamin A, vitamin E,vitamin B12, and the like. As a novel photodynamic medicament, the 5-ALA is not only used for treating local or systemic skin cancer, but also used for diagnosing bladder cancer, digestive tract cancer, lung cancer and the like.
Patent application CN109010366A discloses that 5-aminolevulinic acid (5-ALA) is an endogenous substance, widely exists in tissues and organs of the body, has no photodynamic effect, but can be converted into protoporphyrin (PpIX) with photodynamic effect in vivo, and the metabolic rates of protoporphyrin (PpIX) in normal cells and tumor cells are obviously different, so that the tumor cells can generate characteristic red fluorescence when receiving excitation light, thereby identifying the tumor tissue location.
Disclosure of Invention
The present invention aims to accurately detect circulating tumor cells in peripheral blood. 5-aminoacetic acid (5-ALA) and the enriched tumor circulating cells are cultured in vitro, the tumor cells can generate specific metabolite protoporphyrin (PpIX) in the culture process, the protoporphyrin (PpIX) in cytoplasm of the cells can generate red fluorescence at the wavelength of 635nm, and the tumor circulating cells can be counted by a fluorescence microscope by utilizing the characteristic.
In order to achieve the above object:
the invention firstly relates to the application of 5-ALA in preparing circulating tumor cell detection reagent.
The invention also relates to a circulating tumor cell detection reagent which contains 5-ALA.
The invention also relates to a circulating tumor cell detection kit, which comprises a nucleated cell separation tube, a cell culture tube and a cell culture solution, wherein,
separating liquid with density of 1.077-1.082, organic separating gel with density of 1.085 and anticoagulant for blood are sequentially added into the nucleated cell separating tube,
the cell culture medium comprises 5-ALA and basal cell culture medium.
The circulating tumor cell detection kit also has the following optimized structure:
the circulating tumor cell detection kit also comprises a cell diluent and a cell washing solution.
The blood anticoagulant is selected from sodium citrate, dipotassium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, lithium heparin or sodium heparin.
The nucleated cell separating medium is Ficoll separating medium which consists of a), Ficoll400 and b), diatrizoate sodium.
The organic separation gel includes an organogelling agent, a thixotropic agent, and a density adjusting agent.
The basic cell culture solution adopts RPMI1640 culture solution or M199 culture solution.
The invention also relates to a circulating tumor cell detection method, which adopts the circulating tumor cell detection kit for detection and comprises the following steps:
adding blood sample into nucleated cell separation tube, mixing with anticoagulant, centrifuging under centrifugal force of 1700g-1900g for 15-20 min, standing for layering, placing red blood cells in lower layer of test tube, placing organic gel separation layer and then separation liquid layer on the upper layer, placing platelet-containing plasma component on the uppermost layer, placing nucleated cells on the upper part of separation liquid layer or partially dispersing in the separation liquid, sucking the separation liquid completely for cell culture,
adding the enriched nucleated cell separating medium and the cell culture medium into a cell culture tube according to the volume ratio of 1:2 to 1:5, culturing for 2 hours at the constant temperature of 37 ℃,
after the completion of the culture, the cell culture tube was centrifuged under a centrifugal force of 700g for 5 to 10 minutes, and the centrifuged cell pellet smear was taken and observed under a fluorescence microscope having a wavelength of 635nm to distinguish the nucleated cells whose cytoplasm was purple red, and counted as circulating tumor cells.
The reagent and the method can accurately detect the circulating tumor cells in the peripheral blood. Mainly comprises the enrichment of tumor circulating cells (CTCs), the in vitro culture of the tumor circulating cells (CTCs), and the characterization and counting of the cultured cells.
Detailed Description
The present invention is further illustrated by the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the present invention.
The kit for detecting circulating tumor cells in this embodiment comprises: a nucleated cell separation tube, a cell culture solution, a cell diluent and a cell washing solution.
The nucleated cell separation tube consists of multiple layers of dense media. Before use, first, the nucleated cell separation liquid (density 1.077-1.082) is poured into the bottom of the test tube, then the organic separation gel (density 1.085) with certain viscosity is placed on the top of the separation liquid, and finally the blood anticoagulant is added. The nucleated cell separating medium is preferably Ficoll separating medium, and is composed of polysucrose (Ficoll 400) and diatrizoate or diatrizoate sodium. The organic separation gel is also called blood separation gel, and is composed of an organic gelling agent, a thixotropic agent, a density regulator, and the like. The blood anticoagulant is preferably sodium citrate, and ethylene diamine tetraacetic acid dipotassium, ethylene diamine tetraacetic acid disodium, heparin lithium, heparin sodium and the like can also be used.
The cell culture solution comprises 5-ALA with a certain concentration and basal cell culture solution, wherein the preferable concentration of 5-ALA is 0.5-1.5g/L, and the basal cell culture solution can be RPMI1640 culture solution or M199 culture solution.
One of the realized circulating tumor cell detection kits comprises the following components:
the nucleated cell separating tube mainly comprises Ficoll separating liquid, organic gel and anticoagulant.
The cell diluent mainly comprises sodium citrate and trypsin.
The cell culture solution mainly comprises 5-ALA and RPMI 1640.
The cell washing solution mainly comprises Tween 20 and PBS buffer solution.
A cell culture tube containing a cell culture solution.
When in use, the blood sample and the anticoagulant are evenly mixed and then centrifuged under the condition of certain centrifugal force (1700 g-1900g, 15-20 minutes), and nucleated cells including tumor circulating cells (CTCs) are firstly enriched together by utilizing different volumes and densities of peripheral blood cells. The densest red blood cells (1.093) are in the lower layer of the tube, above which is the second density organic separation gel (1.085), followed by the nucleated cell separation (1.077-1.082), and the uppermost layer is the platelet-containing plasma fraction. The nucleated cells with the density of 1.075-1.080 are positioned at the upper part of the separating medium or partially dispersed in the separating medium, and the separating medium is intensively sucked for cell culture during material taking.
Adding the enriched nucleated cell separation solution and the cell culture solution into a cell culture tube according to the volume ratio of 1:2 to 1:5, culturing for 2 hours at the constant temperature of 37 ℃, wherein the tumor cells and the normal cells can generate specific metabolite protoporphyrin (PpIX) due to the obvious difference of the metabolic rates in the culture process of the tumor cells and the normal cells and 5-aminoacetic acid (5-ALA), so that cytoplasm of the tumor circulating cells is purple, and red fluorescence is generated at the wavelength of 635 nm.
The cultured cell culture tubes were centrifuged (centrifugal force 700g, 5-10 minutes), and the centrifuged cell pellet smears were taken and observed under a fluorescence microscope having a wavelength of 635nm to distinguish nucleated cells whose cytoplasm was purple red, and counted as Circulating Tumor Cells (CTCs).
Claims (9)
1. An application of 5-ALA in preparing the reagent for detecting the circulating tumor cells.
2. A reagent for detecting circulating tumor cells, which is characterized by comprising 5-ALA.
3. A circulating tumor cell detection kit is characterized by comprising a nucleated cell separation tube, a cell culture tube and a cell culture solution, wherein,
separating liquid with density of 1.077-1.082, organic separating gel with density of 1.085 and anticoagulant for blood are sequentially added into the nucleated cell separating tube,
the cell culture medium comprises 5-ALA and basal cell culture medium.
4. The circulating tumor cell detection kit according to claim 3, further comprising a cell diluent and a cell washing solution.
5. The kit for detecting circulating tumor cells according to claim 3, wherein the blood anticoagulant is selected from the group consisting of sodium citrate, dipotassium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, lithium heparin and sodium heparin.
6. The kit for detecting circulating tumor cells according to claim 3, wherein the nucleated cell-separating medium is a Ficoll-separating medium consisting of a), Ficoll400 and b), diatrizoate sodium.
7. The kit for detecting circulating tumor cells according to claim 3, wherein the organic separation gel comprises an organic gelling agent, a thixotropic agent and a density-adjusting agent.
8. The kit for detecting circulating tumor cells according to claim 3, wherein the culture solution of basal cells is RPMI1640 culture solution or M199 culture solution.
9. A method for detecting circulating tumor cells, which is characterized by adopting the circulating tumor cell detection kit of any one of claims 3 to 8 for detection, and comprises the following steps:
adding a blood sample into a nucleated cell separation tube, uniformly mixing with an anticoagulant, then centrifugally separating for 15-20 minutes under the centrifugal force of 1700g-1900g, standing for layering, wherein red blood cells are positioned at the lower layer of a test tube, an organic separation gel layer is arranged above the red blood cells, a separation liquid layer is arranged below the red blood cells, the uppermost layer is a plasma component containing platelets, the nucleated cells are positioned at the upper part of the separation liquid layer or partially dispersed in the separation liquid, and the separation liquid is completely sucked for cell culture,
Adding the enriched nucleated cell separating medium and the cell culture medium into a cell culture tube according to the volume ratio of 1:2 to 1:5, culturing for 2 hours at the constant temperature of 37 ℃,
after the completion of the culture, the cell culture tube was centrifuged under a centrifugal force of 700g for 5 to 10 minutes, and the centrifuged cell pellet smear was taken and observed under a fluorescence microscope having a wavelength of 635nm to distinguish the nucleated cells whose cytoplasm was purple red, and counted as circulating tumor cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910343600.0A CN111855541A (en) | 2019-04-26 | 2019-04-26 | Circulating tumor cell detection reagent, kit and detection method |
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| CN201910343600.0A CN111855541A (en) | 2019-04-26 | 2019-04-26 | Circulating tumor cell detection reagent, kit and detection method |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105683752A (en) * | 2013-06-21 | 2016-06-15 | 国立大学法人冈山大学 | Method using abnormally-activated-cell detection to test for malignant tumors and abnormally-activated-cell apheresis-therapy apparatus |
| CN106929475A (en) * | 2017-02-21 | 2017-07-07 | 中国人民解放军第二军医大学 | A kind of method that liver cancer circulating tumor cell is counted based on 3D temperature sensitive type biogel drop cultures in vitro |
| CN106939297A (en) * | 2017-02-21 | 2017-07-11 | 中国人民解放军第二军医大学 | A kind of method that liver cancer xenograft tumor models are set up based on liver cancer circulating tumor cell biogel drop culture |
| WO2018131989A1 (en) * | 2017-01-16 | 2018-07-19 | 경희대학교 산학협력단 | Composition for photodynamic therapy using gene expressing fluorescent protein and photosensitizer, and photodynamic therapy method using same |
| CN108611322A (en) * | 2018-05-09 | 2018-10-02 | 邹畅 | Method for building up and the application of breast cancer circulating tumor cell system CTC-3, culture medium and CTC-3 |
| CN109207428A (en) * | 2018-09-12 | 2019-01-15 | 上海易对医生物医药科技有限公司 | The separation of circulating tumor cell and cultural method |
-
2019
- 2019-04-26 CN CN201910343600.0A patent/CN111855541A/en not_active Withdrawn
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105683752A (en) * | 2013-06-21 | 2016-06-15 | 国立大学法人冈山大学 | Method using abnormally-activated-cell detection to test for malignant tumors and abnormally-activated-cell apheresis-therapy apparatus |
| WO2018131989A1 (en) * | 2017-01-16 | 2018-07-19 | 경희대학교 산학협력단 | Composition for photodynamic therapy using gene expressing fluorescent protein and photosensitizer, and photodynamic therapy method using same |
| CN106929475A (en) * | 2017-02-21 | 2017-07-07 | 中国人民解放军第二军医大学 | A kind of method that liver cancer circulating tumor cell is counted based on 3D temperature sensitive type biogel drop cultures in vitro |
| CN106939297A (en) * | 2017-02-21 | 2017-07-11 | 中国人民解放军第二军医大学 | A kind of method that liver cancer xenograft tumor models are set up based on liver cancer circulating tumor cell biogel drop culture |
| CN108611322A (en) * | 2018-05-09 | 2018-10-02 | 邹畅 | Method for building up and the application of breast cancer circulating tumor cell system CTC-3, culture medium and CTC-3 |
| CN109207428A (en) * | 2018-09-12 | 2019-01-15 | 上海易对医生物医药科技有限公司 | The separation of circulating tumor cell and cultural method |
Non-Patent Citations (1)
| Title |
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| SATOSHI MATSUSAKA 等: "A novel detection strategy for living circulating tumor cells using 5-aminolevulinic acid", CANCER LETTERS, vol. 355, no. 1, pages 113 - 120, XP029073648, DOI: 10.1016/j.canlet.2014.09.009 * |
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