CN103908306A - Circulating tumor cell in-vivo capturing device and circulating tumor cell in-vivo capturing method on basis of indwelling needles - Google Patents
Circulating tumor cell in-vivo capturing device and circulating tumor cell in-vivo capturing method on basis of indwelling needles Download PDFInfo
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- CN103908306A CN103908306A CN201410153597.3A CN201410153597A CN103908306A CN 103908306 A CN103908306 A CN 103908306A CN 201410153597 A CN201410153597 A CN 201410153597A CN 103908306 A CN103908306 A CN 103908306A
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- circulating tumor
- tumor cell
- remaining needle
- acquisition equipment
- body acquisition
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Abstract
The invention discloses a circulating tumor cell in-vivo capturing device on the basis of indwelling needles. The circulating tumor cell in-vivo capturing device comprises the indwelling needles. Materials capable of adsorbing proteins wrap the outer surfaces of the indwelling needles, and epithelial cell specific antibodies which are used for recognizing specificity and capturing circulating tumor cells are adsorbed on the materials capable of adsorbing the proteins. The invention further discloses a method for capturing the circulating tumor cells by the aid of the capturing device. The circulating tumor cell in-vivo capturing device and the method have the advantages that patients are transfused through the indwelling needles, so that the circulating tumor cells in the blood vessels of the patients can be captured by the modified specific antibodies on the indwelling needles during transfusion; the indwelling needles are pulled out of the patients after the transfusion is completed, so that the captured circulating tumor cells can be removed from the bodies of the patients, and are eluted by eluent; the eluted cells can be observed and counted under microscopes, or immunostaining can be carried out on target molecules to be tested in the cells, and the target molecules to be tested in the cells can be observed through confocal microscopes; the circulating tumor cell in-vivo capturing method is simple in operation, and the target cells can be efficiently and selectively captured by the aid of the circulating tumor cell in-vivo capturing method.
Description
Technical field
The present invention relates to a kind of circulating tumor cell based on remaining needle in body acquisition equipment and method.
Background technology
People just have realized that cancer metastasis is because cancer in situ is sent out cancerous cell in blood to a great extent, is carried into other organs by blood, thereby forms new tumor decades ago.At present, be used as a kind of new liquid diagnostic method for catching of circulating tumor cell (circulating tumor cells, CTCs) with detecting, for following the trail of the diffusion of cancer or the prognosis effect of predicted treatment.Up to now, nearly all to catch with the method detecting about CTCs be all based on get some ml samples from blood, and then CTCs in this sample of Acquisition Detection.The basic law sampling in analytical chemistry is that sample is representative, and sending out of CTCs is irregular, or have a cell and send out in blood, therefore in blood, the distribution of CTCs is inhomogeneous.This will certainly cause the sample of getting from blood is at random not representative, thereby can cause this type of detection method based on sampling very likely to occur false negative.Be that the detection method that toilet is set up can detect 1 cell in 1 milliliter of blood, with respect to the uneven distribution of CTCs in systemic blood, this neither one result accurately.Therefore, being necessary to research and develop one catches and detection method more accurately and effectively.
Summary of the invention
Catch and the present Research of detection method about CTCs for above-mentioned, the invention provides a kind of circulating tumor cell based on remaining needle in body acquisition equipment and method.Can be used for CTCs to realize more accurately and effectively and detecting.
The present invention is achieved by the following technical solutions:
A kind of circulating tumor cell based on remaining needle is at body acquisition equipment, comprise remaining needle, remaining needle outer surface is coated with the material of adsorbable albumen, and on the material of adsorbable albumen, absorption is useful on specific recognition and the epithelial cell specific antibody of catching circulating tumor cell.
Described remaining needle, referring to can be for transfusion, and all different sizes more than indwelling 1h, the transfusion needle of type in vivo.
The material of described adsorbable albumen, refers to the material that can pass through the adsorbed proteins such as covalent coupling, hydrogen bond action, hydrophobic interaction or intermolecular force, preferably polydimethylsiloxane (Poly (dimethylsiloxane), PDMS).The material of adsorbable albumen can physical attachment at remaining needle outer surface.
Described epithelial cell specific antibody, comprises all antibody types for circulating tumor cell surface specific antigen, can be polyclonal antibody, monoclonal antibody or recombinant antibodies, and aptamer.
Described epithelial cell specific antibody, the target tumor of catching as required and selecting, such as: need to catch breast cancer cell time, can select anti-epithelial cell adhesion molecule (Epithelial cell adhesion molecule, EpCAM) antibody.
The described circulating tumor cell based on remaining needle in the preparation method of body acquisition equipment is:
(1) PDMS is coated: body fluid and firming agent thereof before PDMS (before PDMS, body fluid and firming agent thereof are all existing conventional products in prior art) 5:1 ratio mix homogeneously by volume, obtain mixed liquor, then remaining needle is immersed in this mixed liquor, the remaining needle that turns clockwise more than 1 week makes evenly coated, then take out, be placed in 65 DEG C of drying baker and solidify 3 hours, must be coated with the remaining needle of PDMS;
(2) absorption of antibody: it is 1～500 μ gmL that the remaining needle that is coated with PDMS is immersed in to concentration
-1(preferably 100 μ gmL
-1) in epithelial cell specific antibody solution, jolting 2 hours (object is that epithelial cell specific antibody is fully adsorbed on PDMS) gently, by PBS buffer solution for cleaning 3 times, is placed under 4 DEG C of environment and preserves after taking out, stand-by.
Utilize the above-mentioned circulating tumor cell based on remaining needle to catch the method for circulating tumor cell at body acquisition equipment: when the circulating tumor cell of utilization based on remaining needle infused to the cancer patient who carries target circulation tumor cell at body acquisition equipment, in infusion process, the specific antibody that circulating tumor cell in cancer patient's blood vessel is modified on remaining needle is caught; After infusion process completes, remaining needle is extracted, can the circulating tumor cell of catching be cleared out of external, use eluent eluting, circulating tumor cell can be washed down, the cell eluting can be placed under microscope (optical microscope or laser confocal microscope) and observe and count, or does immunostaining for target molecule to be measured in cell, Laser Scanning Confocal Microscope observation.
Described eluent, is selected from phosphate buffered solution (pH7.4,0.1molL
-1), glycine buffer solution (pH2.7,0.1molL
-1) or sodium hydroxide solution (pH13.6,0.4molL
-1).
Circulating tumor cell based on remaining needle of the present invention is in body acquisition equipment and method, and remaining needle adopts epithelial cell specific antibody to modify, and can realize catching target cell high selectivity and high specific.When this kind of clinical employing passes through the retention needle infusion of modifying, can realize catching circulating tumor cell simultaneously.After having infused, when remaining needle is extracted, can the circulating tumor cell of catching be cleared out of external.By after the circulating tumor cell eluting on remaining needle, can carry out to tumor cell the detection (early diagnosis, therapeutic response etc.) of various objects in vitro.Circulating tumor cell based on remaining needle of the present invention, at body catching method, without blood drawing, can not cause any additional injury to human body, with low cost and efficient, simple to operate, can be used for the prediction of cancer patient's early diagnosis and therapeutic response.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1
A kind of circulating tumor cell based on remaining needle is at body acquisition equipment, comprise remaining needle, remaining needle outer surface is coated with PDMS, the upper absorption of PDMS is useful on specific recognition, and (target circulation tumor cell is MCF-7 cell with the epithelial cell specific antibody of catching circulating tumor cell, selected epithelial cell specific antibody is anti-EpCAM antibody, purchased from Beijing Bo Aosen Bioisystech Co., Ltd).
The specifications and models of described remaining needle are 24G × 0.75 ＂, 0.7mm × 19mm.
The described circulating tumor cell based on remaining needle in the preparation method of body acquisition equipment is:
(1) PDMS is coated: body fluid and firming agent 5:1 ratio mix homogeneously by volume thereof before PDMS, obtain mixed liquor, then remaining needle is immersed in this mixed liquor, the remaining needle that turns clockwise makes evenly coated, then take out, be placed in 65 DEG C of drying baker and solidify 3 hours, must be coated with the remaining needle of PDMS;
(2) absorption of antibody: the remaining needle that is coated with PDMS is immersed in to 100 μ gmL
-1epithelial cell specific antibody solution in, jolting 2 hours gently, by PBS buffer solution for cleaning 3 times, is placed under 4 DEG C of environment and preserves after taking out, stand-by.
Test example: choose the laboratory animal that two kinds of animals of rabbit and mice catch at body as circulating tumor cell, taking the breast carcinoma cell strain of can commercialization buying---MCF-7 is as example, and checking is at the effectiveness of body catching method.
In order to investigate animal immune system to injecting the scavenging action of MCF-7 cell, choose the nude mice of immunodeficiency for catching experiment at body.By the solution that contains certain MCF-7 cell number (specifically in table 1) preparing, inject respectively the left ear auricular vein of rabbit, in mice, vein of nude mice afterbody, remaining needle through modifying prepared by embodiment 1 thrusts rabbit auris dextra auricular vein, in mice, another vein of nude mice afterbody, after 3 hours, remaining needle is taken out, with pH be 2.7, concentration is 0.1molL
-1gly-HCl solution the MCF-7 cell of catching is eluted in the hole of ELISA Plate from remaining needle, be placed under microscope and observe, counting cells number.
Table 1 has been added up cell number and the capture rate of actual acquisition while adding different cell number.From table 1, injecting the MCF-7 cell of the different numbers of rabbit, mice and nude mice all can successfully be caught, due to remaining needle surface area and the adsorption capacity restriction to cell, capture rate reduces with adding cell number increase, because the circulating tumor cell number in patient blood is considerably less, therefore,, when the method is used for Clinical detection, the reduction of this capture rate can not produce substantial impact.Because nude mice has immunodeficiency, the MCF-7 that injects nude mice vein can not destroyed by immune system, and therefore remaining needle is for the capture rate of nude mice far above wild mouse, and this has also further verified the feasibility of this method.Because remaining needle finishing has antibody, play the sealing process to PDMS adsorption site, can greatly reduce non-specific adsorption, experiment discovery, modified remaining needle can not adsorb more erythrocyte and leukocyte than the remaining needle of and antibody absorption coated without PDMS.
Table 1
Claims (9)
1. the circulating tumor cell based on remaining needle is at body acquisition equipment, it is characterized in that: comprise remaining needle, remaining needle outer surface is coated with the material of adsorbable albumen, and on the material of adsorbable albumen, absorption is useful on specific recognition and the epithelial cell specific antibody of catching circulating tumor cell.
2. the circulating tumor cell based on remaining needle according to claim 1, at body acquisition equipment, is characterized in that: the material of described adsorbable albumen refers to the material that can pass through covalent coupling, hydrogen bond action, hydrophobic interaction or intermolecular force adsorbed proteins.
3. the circulating tumor cell based on remaining needle according to claim 1, at body acquisition equipment, is characterized in that: the material of described adsorbable albumen is selected from polydimethylsiloxane.
4. the circulating tumor cell based on remaining needle according to claim 1, at body acquisition equipment, is characterized in that: described epithelial cell specific antibody is selected from polyclonal antibody, monoclonal antibody, recombinant antibodies or aptamer.
5. the circulating tumor cell based on remaining needle according to claim 1, at body acquisition equipment, is characterized in that: the target tumor that described epithelial cell specific antibody is caught as required and selecting.
6. the circulating tumor cell based on remaining needle according to claim 1, at body acquisition equipment, is characterized in that: need to catch breast cancer cell time, described epithelial cell specific antibody is selected from anti-epithelial cell adhesion molecule antibody.
7. in claim 1～6, the circulating tumor cell based on remaining needle described in any one, in the preparation method of body acquisition equipment, is characterized in that: step is as follows:
(1) PDMS is coated: body fluid and firming agent 5:1 ratio mix homogeneously by volume thereof before PDMS, obtain mixed liquor, then remaining needle is immersed in this mixed liquor, the remaining needle that turns clockwise more than 1 week makes evenly coated, then take out, be placed in 65 DEG C of drying baker and solidify 3 hours, must be coated with the remaining needle of PDMS;
(2) absorption of antibody: it is 1～500 μ gmL that the remaining needle that is coated with PDMS is immersed in to concentration
-1epithelial cell specific antibody solution in, jolting 2 hours, by PBS buffer solution for cleaning 3 times, to obtain final product after taking out.
8. utilize the circulating tumor cell based on remaining needle described in any one in claim 1～6 to catch the method for circulating tumor cell at body acquisition equipment, it is characterized in that: utilize the circulating tumor cell based on remaining needle to infuse to the cancer patient who carries target circulation tumor cell at body acquisition equipment, in infusion process, the specific antibody that circulating tumor cell in cancer patient's blood vessel is modified on remaining needle is caught; After infusion process completes, remaining needle is extracted, thereby the circulating tumor cell of catching is cleared out of external, use eluent eluting, circulating tumor cell is washed down.
9. method according to claim 8, is characterized in that: described eluent is selected from phosphate buffered solution (pH7.4,0.1molL
-1), glycine buffer solution (pH2.7,0.1molL
-1) or sodium hydroxide solution (pH13.6,0.4molL
-1).
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Cited By (8)
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| WO2017044723A1 (en) * | 2015-09-09 | 2017-03-16 | Memorial Sloan Kettering Cancer Center | Capture device for detection of malignant cells in blood and methods thereof |
| CN107625534A (en) * | 2016-07-18 | 2018-01-26 | 上海千山医疗科技有限公司 | Indwelling needle assembly and the circulating tumor cell supporting with remaining needle are in body acquisition equipment |
| CN108531455A (en) * | 2018-03-09 | 2018-09-14 | 大连理工大学 | Polyphenol coating for circulating tumor cell capture |
| CN108761080A (en) * | 2018-08-16 | 2018-11-06 | 中国科学院合肥物质科学研究院 | One specific cells in body captures box |
| CN109991418A (en) * | 2019-04-18 | 2019-07-09 | 山东师范大学 | A kind of circulating tumor cell capturing device and method |
| US10987037B2 (en) | 2003-12-22 | 2021-04-27 | John Wayne Cancer Institute | Method and apparatus for in vivo surveillance of circulating biological components |
| WO2021120209A1 (en) * | 2019-12-18 | 2021-06-24 | 深圳先进技术研究院 | In-vivo capture system and method for cell to be tested |
| US11160542B2 (en) | 2016-06-09 | 2021-11-02 | Haimachek, Inc. | Collector for detection and reversible capturing of cells from body fluids in vivo |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10987037B2 (en) | 2003-12-22 | 2021-04-27 | John Wayne Cancer Institute | Method and apparatus for in vivo surveillance of circulating biological components |
| WO2017044723A1 (en) * | 2015-09-09 | 2017-03-16 | Memorial Sloan Kettering Cancer Center | Capture device for detection of malignant cells in blood and methods thereof |
| US11160542B2 (en) | 2016-06-09 | 2021-11-02 | Haimachek, Inc. | Collector for detection and reversible capturing of cells from body fluids in vivo |
| CN107625534A (en) * | 2016-07-18 | 2018-01-26 | 上海千山医疗科技有限公司 | Indwelling needle assembly and the circulating tumor cell supporting with remaining needle are in body acquisition equipment |
| CN108531455A (en) * | 2018-03-09 | 2018-09-14 | 大连理工大学 | Polyphenol coating for circulating tumor cell capture |
| CN108761080A (en) * | 2018-08-16 | 2018-11-06 | 中国科学院合肥物质科学研究院 | One specific cells in body captures box |
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| WO2021120209A1 (en) * | 2019-12-18 | 2021-06-24 | 深圳先进技术研究院 | In-vivo capture system and method for cell to be tested |
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Application publication date: 20140709 |