CN108690832A - The capture of circulating tumor cell and method for releasing - Google Patents
The capture of circulating tumor cell and method for releasing Download PDFInfo
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- CN108690832A CN108690832A CN201710236512.1A CN201710236512A CN108690832A CN 108690832 A CN108690832 A CN 108690832A CN 201710236512 A CN201710236512 A CN 201710236512A CN 108690832 A CN108690832 A CN 108690832A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
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Abstract
The invention discloses a kind of capture of circulating tumor cell and method for releasing, include by can digestion tumour-specific combine the peptide modified runner to micro-fluidic chip in;It is passed through the blood sample containing tumour cell in the micro-fluidic chip runner, carries out the capture of tumour cell;Be passed through in the micro-fluidic chip runner selected enzyme to this can the tumour-specific combination polypeptide of digestion shear, realize the release to captured cell.The present invention is based on PDMS micro-fluidic chips, by modifying the tumour cell target polypeptide can be sheared by certain enzyme in runner, realize the capture and release to circulating tumor cell, compared to antibody capture circulating tumor cell, can digestion tumour-specific combination polypeptide it is of low cost, and the release after capture can be completed, cell is further cultured for convenient for the later stage and is further studied, production application is suitble to.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of circulating tumor cell in circulating tumor cell detection field
Capture and method for releasing.
Background technology
Tumour is one of the significant threat that the whole world faces, at present still without the effective means for thoroughly effecting a radical cure tumour, only
Accomplish early detection, it is early to diagnose, it can just accomplish effective treatment of tumour.The detection of circulating tumor cell (CTCs) can be answered effectively
Play the role of assessment for the early diagnosis of metastatic tumour, and to therapeutic effect, but quantity of the CTCs in peripheral blood is few,
There are about 1-10 circulating tumor cells in every 10 milliliters in the peripheral blood of cancer patient.In current existing detection circulating tumor
In the method for cell, most of simple sensitive detection methods all rely on the knowledge of the EpCAM to circulating tumor cell surface
Not, it is detected be EpCAM expression tumour cell.But it is this dependent on EpCAM, for the CTCs of Epithelial
Detection method has some limitations.First, in the development and transfer process of tumour, part cell can occur to fill between epithelium
Matter converts (EMT), and the catching method dependent on EpCAM can not effectively detect the CTCs that EMT has occurred in this part, to make
The missing inspection of pairs of CTCs, and lead to a degree of false negative;The use cost of second, EpCAM antibody is high, limits its life
Production and application;The capture of third, antibody is only capable of detecting the presence of CTCs, can not go deep into CTCs in small runner
Research detection, therefore how from runner discharge CTCs be also an important issue project.
Currently, having correlative study passes through the antibody or aptamer reality for circulating tumor cell surface antigens unique
The capture of existing circulating tumor cell.However whether can catching for circulating tumor cell efficiently be completed by other biological molecule
It obtains, a kind of relatively inexpensive method of searching captures and obtains circulating tumor cell with no damage, and being one is worth that explores to ask
Topic.
Invention content
In view of deficiency in the prior art, the main purpose of the present invention is to provide it is a kind of with can digestion tumour-specific
The method for capturing simultaneously release cycle tumour cell in micro-fluidic chip in conjunction with polypeptide, this method relative inexpensiveness, while energy
Realize the capture and release to circulating tumor cell.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
A kind of capture of circulating tumor cell and method for releasing, include the following steps:
(1) by can digestion tumour-specific combine the peptide modified runner to micro-fluidic chip in;
(2) it is passed through the blood sample containing tumour cell in the micro-fluidic chip runner, carries out catching for tumour cell
It obtains;
(3) be passed through in the micro-fluidic chip runner selected enzyme to it is described can digestion tumour-specific combination polypeptide
It is sheared, realizes the release to captured cell.
In one embodiment, the method for modifying in step (1) is including the use of the parent modified in the micro-fluidic chip runner
And element with it is described can digestion tumour-specific combination polypeptide one end mark biotin between affinity interaction modified,
And/or chemical modification method.
In one embodiment, the chemical modification method is sulfydryl modification.
In one embodiment, it is described can the sequence of tumour-specific combination polypeptide of digestion be
WFCSWYGGDTCVQGGENLYFQGGGK-Biotin;Wherein, targeting sequence is:WFCSWYGGDTCVQ, cleavage sequence are:
ENLYFQ。
In one embodiment, step (2) includes:Using can digestion tumour-specific combination polypeptide to the spy of tumour cell
Anisotropic combination realizes the capture to specific tumors cell.
In one embodiment, further include before step (2):In the micro-fluidic chip after step (1) modification
It is passed through certain density tumor cell suspension in runner and captures the function of circulating tumor cell to verify it.
Further include that pretreated step is carried out to blood sample in one embodiment, before step (2):It is thin tumour will to be contained
The blood sample of born of the same parents carries out erythrocyte splitting so that the circulating tumor cell in the blood sample is enriched to predetermined concentration.
In one embodiment, the blood sample containing tumour cell is subjected to erythrocyte splitting so that the blood sample
In circulating tumor cell be enriched to predetermined concentration and specifically include:Red blood cell is added in the blood sample containing tumour cell to split
Liquid is solved, and is at least repeated once centrifugation, removal supernatant, adds the step of precipitation is resuspended in PBS.
Compared with prior art, beneficial effects of the present invention include:
(1) polypeptide can not only specifically bind tumour cell and realize capture, and pass through design relative to conventional antibodies
Be added can digestion sequence, can realize and nondestructively release the cell of capture, it is of low cost, for deeper into research create
Make possibility.
(2) target polypeptide has diversity to the capture of tumour cell, be not limited to it is described in the present invention can digestion A-1
The design of target polypeptide, restriction enzyme site can also be varied, can by using other target polypeptides and other restriction enzyme sites
The micro-fluidic chip with higher capture rate and sensitivity is further designed, research space is wider, has broader
Foreground.
(3) peptide modified micro-fluidic chip of the invention is easily prepared, of low cost.
Description of the drawings
Fig. 1 be circulating tumor cell of the present invention one embodiment of capture and method for releasing in can digestion tumour-specific knot
Close capture and the release process schematic of peptide modified micro flow chip runner inner tumour cell;
Fig. 2 be circulating tumor cell of the present invention one embodiment of capture and method for releasing in obtain can digestion tomour specific
Property combine peptide modified micro flow chip to the capture ability schematic diagram of tumour cell;
Fig. 3 be circulating tumor cell of the present invention one embodiment of capture and method for releasing in obtain can digestion tomour specific
Property combine in peptide modified micro flow chip enzyme to the releasability schematic diagram of cell.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention is described in further detail, but protection scope of the present invention is not
It is limited to this.
Cooperation referring to Fig.1, introduces the capture of circulating tumor cell of the present invention and a specific implementation mode of method for releasing, packet
Include following steps:
Step S1:Prepare micro-fluidic chip.
In one embodiment, the preparation method of micro-fluidic chip specifically includes:By aggressiveness before PDMS and curing agent 10:1 mixing,
Stir evenly, pour in being carved on the silicon chip mold of runner pattern, control thickness 5mm, be placed in vacuum desiccator vacuumize with
Exclude bubble.90 DEG C of solidification 1h.PDMS blocks are cut out with cutter, are punched in runner exit and import.By clean glass slide and
PDMS blocks are put into PLASMA Cleaner, and it is 500mTorr to be evacuated to air pressure in instrument, are taken after opening PLASMA effects 30s
Go out, PDMS blocks are bonded with glass slide, it is light to press, it is placed in 90 DEG C of bakings of drying box and is allowed to permanently be bonded for 5 minutes.
Step S2:By can digestion tumour-specific combine the peptide modified runner to micro-fluidic chip in.
The method of modifying referred in this step including the use of the Avidin modified in micro-fluidic chip runner with can digestion
Affinity interaction between the biotin of tumour-specific combination polypeptide one end label is modified and/or chemical modification method, into
Exemplarily, which is sulfydryl modification to one step.
In one embodiment, Avidin method of modifying specifically includes in runner:With 10 μ L/min's under the conditions of room temperature is protected from light
Flow velocity is passed through the ethanol solution of 2% (v/v) silane reagent, and being passed through ethanol solution with the flow velocity of 10 μ L/min cleans 20 minutes, with
The flow velocity of 10 μ L/min is passed through deionized water and cleans 30 minutes, and the liquid drying in chip is placed on 110 DEG C of bakings of drying box
60 minutes.Being passed through glutaraldehyde reaction solution 1 hour after chip cooling technique with the flow velocity of 10 μ L/min, (glutaraldehyde reaction solution is prepared:
50% glutaraldehyde 1mL, PBS20mL, sodium cyanoborohydride 40mg, pH 7.4), then deionized water is passed through with the flow velocity of 10 μ L/min
The PBS solution for the Avidin for being passed through 100 μ g/mL after liquid drying in chip is placed in 4 DEG C of preservations by cleaning 30 minutes.Wherein,
The ethanol solution of the silane reagent can be selected from 3- aminopropyl triethoxysilanes.
In one embodiment, can the tumour-specific of digestion combine peptide modified (label biotin) method to specifically include:With
The flow velocity of 10 μ L/min is passed through deionized water in micro-fluidic chip runner and cleans the Avidin that removal in 10 minutes is not coupled, will
1mg/mL it is biotinylated can the PBS solution of tumour-specific combination polypeptide of digestion be passed through in fluid channel, be incubated 45min,
It is passed through PBS cleanings after ten minutes with the flow velocity of 10 μ L/min, is passed through 2% (w/v) BSA solution and closes 1 hour, with 10 μ L/min's
Flow velocity be passed through PBS cleaning be placed in after ten minutes 4 DEG C it is spare.
In one embodiment, can the sequence of tumour-specific combination polypeptide of digestion be
WFCSWYGGDTCVQGGENLYFQGGGK-Biotin;Wherein, targeting sequence is:WFCSWYGGDTCVQ, cleavage sequence are:
ENLYFQ, residue sequence are catenation sequence.
Step S3:Certain density tumour cell is passed through in the micro-fluidic chip runner after step S1 modifications to suspend
Liquid captures the function of circulating tumor cell to verify it.
Step S4:Blood sample is pre-processed:Blood sample containing tumour cell is subjected to erythrocyte splitting, is made
The circulating tumor cell obtained in the blood sample is enriched to predetermined concentration.
This step can specifically include and erythrocyte cracked liquid is added in the blood sample containing tumour cell, and at least heavy
Multiple primary centrifugation, removal supernatant, add the step of precipitation is resuspended in PBS.In one embodiment, the fresh anticoagulations of 1mL is taken (to contain
Tumour cell A549), 10mL erythrocyte cracked liquids are added and (are purchased from TBDscience, article No.:NH4CL2009) gently piping and druming is mixed
It is even, lysis at room temperature 2 minutes;It then centrifuges 5 minutes for 500g4 DEG C, abandons red supernatant;It adds 5mL PBS and precipitation, 500g 4 is resuspended
DEG C centrifugation 3 minutes, abandons supernatant, is repeated 1 times, wash 2 times altogether, cell precipitation is resuspended in 1mL PBS, realizes circulating tumor cell
Enrichment.
Step S5:The above-mentioned blood sample containing tumour cell by enrichment is passed through in the micro-fluidic chip runner
This, carries out the capture of tumour cell.
Using can the tumour-specific combination polypeptide of digestion the specific binding of tumour cell is acted on, may be implemented to spy
Determine the capture of tumour cell.Such as in present embodiment can the tumour-specific combination polypeptide of digestion can to specifically bind people non-
Small cell lung cancer cell A549.Pancreatin digests A549 cells, and PBS is diluted to cell suspending liquid, then is passed through with the flow velocity of 1 μ L/min
In micro-fluidic chip runner, to the cell count in runner under microscope, make have 100-200 cell in every runner;Finally
5min is rinsed to wash away no captured cell with the flow velocity of 1 μ L/min PBS.
As shown in Fig. 2, with another to the uncombined polypeptides of A549 as a contrast, PBS cleaning after compare it is peptide modified
Tumour cell remains little in runner, and in present embodiment can the tumour-specific combination polypeptide of digestion have preferably to A549
Capture acts on.
Step S6:Be passed through in the micro-fluidic chip runner selected enzyme to it is described can digestion tumour-specific combine
Polypeptide is sheared, and realizes the release to captured cell.
In one embodiment, enzyme solutions (being passed through PBS in control chip, other conditions are consistent) are passed through, are made at room temperature
5min is rinsed to discharge captured cell with 15min, then with the flow velocity of 1 μ L/min PBS.To remaining in runner under microscope
Cell count.
As shown in figure 3, PBS is passed through after capture tumour cell in control chip, since the combination of polypeptide is only a small amount of
Cell is rinsed, and tests in chip, and after being passed through enzyme solutions, most tumour cells are released.
It should be noted that the tumour cell referred in the above embodiment, can digestion tumour-specific combination polypeptide
Correspond to each other, for example, in corresponding present embodiment can the tumour cell of tumour-specific combination polypeptide of digestion be A549,
And in other examples, tumour cell and target polypeptide could alternatively be any combinations that can be specifically bound each other,
And cleavage sequence may be any other sequence being digested.
In conclusion the present invention is based on PDMS micro-fluidic chips, by being modified in runner can be cut by certain enzyme
The tumour cell target polypeptide cut realizes capture and release to circulating tumor cell, thin compared to antibody capture circulating tumor
Born of the same parents, can digestion tumour-specific combination polypeptide it is of low cost, and can complete capture after release, convenient for the later stage to cell into
Row is further cultured for and further studies, and has broad application prospects in the detection field of circulating tumor cell.
It should be pointed out that the specific implementation mode of present invention described above, is not intended to limit the scope of the present invention..
Any technique according to the invention design made various other corresponding changes and deformation, should be included in right of the present invention
It is required that protection domain in.
Claims (8)
1. capture and the method for releasing of a kind of circulating tumor cell, it is characterised in that include the following steps:
(1) by can digestion tumour-specific combine the peptide modified runner to micro-fluidic chip in;
(2) it is passed through the blood sample containing tumour cell in the micro-fluidic chip runner, carries out the capture of tumour cell;
(3) be passed through in the micro-fluidic chip runner selected enzyme to it is described can digestion tumour-specific combination polypeptide carry out
The release to captured cell is realized in shearing.
2. capture and the method for releasing of circulating tumor cell according to claim 1, which is characterized in that in step (1)
Method of modifying including the use of the Avidin modified in the micro-fluidic chip runner with it is described can the tumour-specific of digestion combined
Affinity interaction between the biotin of polypeptide one end label is modified and/or chemical modification method.
3. capture and the method for releasing of circulating tumor cell according to claim 2, which is characterized in that the chemical modification
Method is sulfydryl modification.
4. capture and the method for releasing of circulating tumor cell according to claim 1, which is characterized in that it is described can digestion
The sequence of tumour-specific combination polypeptide is WFCSWYGGDTCVQGGENLYFQGGGK-Biotin;Wherein, targeting sequence is:
WFCSWYGGDTCVQ, cleavage sequence are:ENLYFQ.
5. capture and the method for releasing of circulating tumor cell according to claim 1, which is characterized in that step (2) includes:
Using can the tumour-specific combination polypeptide of digestion the specific binding of tumour cell is acted on, realize to specific tumors cell
Capture.
6. capture and the method for releasing of circulating tumor cell according to claim 1, which is characterized in that step (2) it
Before further include:Certain density tumour cell is passed through in the micro-fluidic chip runner after step (1) modification to suspend
Liquid captures the function of circulating tumor cell to verify it.
7. capture and the method for releasing of circulating tumor cell according to claim 1, which is characterized in that before step (2)
Further include that pretreated step is carried out to blood sample:Blood sample containing tumour cell is subjected to erythrocyte splitting so that
Circulating tumor cell in the blood sample is enriched to predetermined concentration.
8. capture and the method for releasing of circulating tumor cell according to claim 7, which is characterized in that it is thin tumour will to be contained
The blood sample of born of the same parents carries out erythrocyte splitting so that it is specific that the circulating tumor cell in the blood sample is enriched to predetermined concentration
Including:Erythrocyte cracked liquid is added in the blood sample containing tumour cell, and is at least repeated once centrifugation, removal supernatant
Liquid adds the step of precipitation is resuspended in PBS.
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Cited By (6)
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| CN110643482A (en) * | 2019-08-27 | 2020-01-03 | 宁波美晶医疗技术有限公司 | Preparation method and application of nano-structure surface chip for capturing and releasing circulating tumor cells |
| CN112924363A (en) * | 2021-01-22 | 2021-06-08 | 中国科学院苏州纳米技术与纳米仿生研究所 | Intermediate circulating tumor cell as tumor diagnosis and prognosis marker and application thereof |
| CN113713866A (en) * | 2021-08-05 | 2021-11-30 | 武汉大学 | Human bone microfluidic chip embedded in cancer cell membrane, preparation method thereof and application of chip in separation of circulating tumor cells |
| CN114249834A (en) * | 2021-12-23 | 2022-03-29 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chimeric antigen receptor capable of specifically targeting tumor cells, expression gene thereof, NK cell modified by chimeric antigen receptor and application |
| CN114712521A (en) * | 2022-03-22 | 2022-07-08 | 郑州大学 | CD44 receptor-targeted drug, and preparation method and application thereof |
| CN114874989A (en) * | 2022-04-11 | 2022-08-09 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for capturing circulating tumor cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643482A (en) * | 2019-08-27 | 2020-01-03 | 宁波美晶医疗技术有限公司 | Preparation method and application of nano-structure surface chip for capturing and releasing circulating tumor cells |
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| CN113713866A (en) * | 2021-08-05 | 2021-11-30 | 武汉大学 | Human bone microfluidic chip embedded in cancer cell membrane, preparation method thereof and application of chip in separation of circulating tumor cells |
| CN114249834A (en) * | 2021-12-23 | 2022-03-29 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chimeric antigen receptor capable of specifically targeting tumor cells, expression gene thereof, NK cell modified by chimeric antigen receptor and application |
| CN114249834B (en) * | 2021-12-23 | 2023-06-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | Chimeric antigen receptor capable of specifically targeting tumor cells, expressed gene thereof, modified NK cells and application thereof |
| CN114712521A (en) * | 2022-03-22 | 2022-07-08 | 郑州大学 | CD44 receptor-targeted drug, and preparation method and application thereof |
| CN114874989A (en) * | 2022-04-11 | 2022-08-09 | 中国科学院苏州纳米技术与纳米仿生研究所 | Method for capturing circulating tumor cells |
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