CN103923192B - For polypeptide and the application thereof of enrichment, separation and detection circulating tumor cell - Google Patents
For polypeptide and the application thereof of enrichment, separation and detection circulating tumor cell Download PDFInfo
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- CN103923192B CN103923192B CN201310013908.1A CN201310013908A CN103923192B CN 103923192 B CN103923192 B CN 103923192B CN 201310013908 A CN201310013908 A CN 201310013908A CN 103923192 B CN103923192 B CN 103923192B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
- C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
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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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The invention provides a kind of polypeptide for enrichment, separation and detection circulating tumor cell and application thereof, does is the aminoacid sequence of described polypeptide SEQ? ID? aminoacid sequence shown in NO:1; Also comprise polypeptide-magnetic nanoparticle, it contains magnetic nanoparticle and the polypeptide for specific recognition tumour cell described above with its phase coupling; And described polypeptide or polypeptide-magnetic nanoparticle are for the preparation of enrichment, separation or the product detecting circulating tumor cell or the application be used for the treatment of in the medicine of metastases relative disease.The present invention proposes a kind of novel method utilizing polypeptide cells surface antigen to carry out specific recognition, and invented the polypeptide-magnetic nanoparticle material of a kind of new enrichment, separation and detection CTC, provide a kind of enrichment, separation and detection CTC quick, accurately and the detection technique of low cost, for the diagnosis of clinical tumor transfer patient, Treatment and Prognosis provide effective ways.
Description
Technical field
The invention belongs to biomedicine technical field, relate to a peptide species, particularly relate to one and may be used for enrichment, separation and detection circulating tumor cell (CirculatingTumorCell, CTC) polypeptide-magnetic nanoparticle and its preparation method and application, the method accurately, fast, greatly reduce the cost of CTC cell of enrichment, the separation and detection Epithelial Cell Adhesion factor (EpithelialCellAdhesionMolecule, EpCAM) high expression level.
Background technology
Cancer is one of major disease threatening the mankind, invasion inhibition is one of the most significant feature of malignant tumour, tumour cell is spontaneous or come off from primary tumo(u)r because of operation of diagnosis and treatment, there is epithelial-mesenchymal and transform (Epithelial-MesenchymalTransition, EMT), thus there is flow characteristics, enter in peripheral blood, just define circulating tumor cell (CTC).Therefore in peripheral blood, detect that CTC imply that likely metastases occurs, in addition, the current research of Harvard Medical School of the U.S. shows, tumor in situ tissue can attract CTC cell again to infiltrate in primary tumo(u)r thus promote that the growth of tumour (can with reference to people such as K.Pantel, Nat.Rev.Clin.Oncol.2009,6,339-351; The people such as J.Kaiser, Science2010,327,1072-1074), therefore the efficient detection of CTC has directive significance for research mechanism of tumor metastasis, and to instructing oncotherapy, judging result for the treatment of, inferring that prognosis provides reliable reference, very important meaning can be had to the transfer or recurrence of monitoring tumour.
Since Australian doctor ThomasAshworth in 1869 observes CTC under the microscope first from the blood of a male tumor transfer patient, the research of CTC causes the extensive concern of people.Few (the about several CTCs/10 of CTCs content in blood
5~ 10
7individual mononuclearcell), people have carried out constantly probing into the enrichment detecting method of CTC.These methods can be divided into two classes: mechanical process and immune partition method (can with reference to P.Paterlini-Brechot & N.L.Benali, CancerLett.2007,253,180-204).The former be according to CTC and the white corpuscle in blood and erythrocytic cell dia and cell density different, by filter or the method for density gradient centrifugation realizes; Latter mainly utilizes some specific marker things on CTC surface as common epithelial marker thing cytokeratin (Cytokeratins, CKs), the Epithelial Cell Adhesion factor (EpCAM), tumour embryonal antigen (CarcinoEmbryonicAntigene, and human epidermal growth factor receptor 2 (HumanEpidermalGrowthFactorReceptor2 CEA), HER2) etc., realizing CTC by antigen and antibody specific association reaction (can with reference to people such as H.Xu with being separated of other cells in blood, Biomaterials2011,32,9758-9765; The people such as S.L.Stott, Proc.Natl.Acad.Sci.USA2010,107,18392-18397; The people such as S.Nagrath, Nature2007,450,1235-1239; The people such as A.-E.Saliba, Proc.Natl.Acad.Sci.USA2010,107,14524-14529; The people such as X.Wang, CancerRes.2011,71,1526-1532).
Compared with immune separation method, at the bottom of mechanical separation method sensitivity, the poor specificity of separation.Therefore people update immune isolation technique, attempt to improve separation efficiency.That the first is widely used for detection CTC is CellSearch, and it is by wrapping the identification being realized the tumour cell to epithelium source by anti-EpCAM antibody on magnetic bead, as mammary cancer and colon cancer cell.CellSearch is successfully used for evaluating tumor prognosis effect clinically, is used as the Prognosis method of Metastasis in Breast Cancer in 2004 by U.S. FDA approval; In order to improve the accuracy that CTCs detects, Massachusetts General Hospital (MGH) the team new device of a kind of CTCChip of being called detects CTC, CTCChip has 78000 small bags by the action site of anti-EpCAM antibody, like this when blood flow is out-of-date, only have tumour cell identified, MGH team delivers in 2007 for the clinical test results of hepatoma Metastasis patient on Nature.Thereafter, this group is collected the CTC of patients with lung cancer with CTCChip again and has been carried out genetic analysis to it.Recently, AndrewWang seminar reports for work and greatly to improve by the sorting method efficiency of the efficiency ratio microballoon of the nano-sized iron oxide grain sorting CTCs of antibody bag quilt, and maximum sorting index reaches 84%.
In these immunoassays, the Epithelial Cell Adhesion factor (EpCAM) and CKs are the conventional surface markers that CTC detects.But, due to the restriction of antibody producing cost and production technology, cause this technical costs based on EpCAM and CKs antibody test CTC higher, need about 600 dollars as CellSearch detects a blood sample, therefore finding can have important meaning with the novel compound of the selectively targeted combination of EpCAM and CKs.
In recent years, the identification that investigators are utilizing polypeptide to carry out tumour cell, aspect, targets neoplastic cells surface expands to be studied widely, people filter out the polypeptide compound distinguishing the different action site of the different tumour cell of target in a large number from polypeptide that is natural and that synthesize (can with reference to M.Shadidi & M.Sioud, FASEBJ.2003,17,256-258; M.Shadidi & M.Sioud, DrugResist.Updates2003,6,363-371).For the detection of the CTC of high metastatic tumour, the polypeptide of development specific recognition EpCAM, CKs has very important significance.
Summary of the invention
The object of the invention is for detecting at present the high deficiency of CTC cost, a kind of enrichment, the new approaches of separation and detection CTC and novel method are provided.An object of the present invention is to design and the polypeptide fragment of specific recognition CTC can substitute existing anti-CTC antibody, thus reduce testing cost.Another object of the present invention is in conjunction with existing nanotechnology, by specific recognition polypeptide and nano particle coupling, namely provides one peptide species-nano particle, improves the detection efficiency to CTC rare in blood.Another object of the present invention is to provide the purposes of specific recognition polypeptide-nano particle in the transfer or recurrent tumor treatment effectiveness evaluation of monitoring tumour.
Except as otherwise noted, " polypeptide-magnetic nanoparticle " herein refers to " polypeptide is at the magnetic nanoparticle of finishing ".
Except as otherwise noted, " 2E7,2E8,2E9 " herein represents that " negative cells number is 2 × 10 respectively
7individual, 2 × 10
8individual, and 2 × 10
9individual ".
Except as otherwise noted, " combination rate " herein refers to " avtive spot of a magnetic nanoparticle having been modified how many polypeptide ".
Except as otherwise noted, " separation rate " herein refers to " ratio that the number of the cell sample of separation accounts for the cell sample number added ".
For above-mentioned purpose, technical scheme of the present invention is as follows:
On the one hand, the invention provides a kind of polypeptide for specific recognition circulating tumor cell, the aminoacid sequence of described polypeptide is the aminoacid sequence shown in following SEQIDNO:1:
VRRDAPRFSMQGLDACGGNX
1X
2(X
3)
n1(X
4)
n2
Wherein, X
1for Cys or Asn;
X
2for Cys or Asn;
X
3for Asn, n1=0 or 1;
X
4for Asn, n2=0 or 1.
Preferably, the aminoacid sequence of described polypeptide is selected from the aminoacid sequence shown in SEQIDNO:2 and SEQIDNO:3, described polypeptide identifies circulating tumor cell by specific binding cell epithelia adhesion factor (EpithelialCellAdhesionMolecule, EpCAM).
On the other hand, the invention provides one peptide species-magnetic nanoparticle, its contain magnetic nanoparticle and with the polypeptide for specific recognition circulating tumor cell described in the invention described above of its phase coupling.
Preferably, the mass ratio of described polypeptide and magnetic nanoparticle is 0.01:5 ~ 4:5.
Preferably, the mass ratio of described polypeptide and magnetic nanoparticle is 1:5 ~ 3:5.
Preferably, described magnetic nanoparticle is the magnetic nanoparticle that streptavidin is modified.
Preferably, the particle diameter of described magnetic nanoparticle is 600 ~ 1500nm.
Preferably, the particle diameter of described magnetic nanoparticle is 800nm.
Also on the one hand, the invention provides the preparation method of the polypeptide-magnetic nanoparticle for specific recognition circulating tumor cell described in a kind of the invention described above, described method comprises the steps:
1) polypeptide for specific recognition circulating tumor cell described in the invention described above is prepared;
2) polypeptide in step 1) is made the polypeptide solution of biotin modification;
3) magnetic nanoparticle that streptavidin is modified is dissolved in damping fluid and makes solution, preferably, also comprise the step of being undertaken by method that is ultrasonic or vortex by solution of magnetic nanoparticles disperseing;
4) by step 2) in after the polypeptide solution of biotin modification mixes with the solution of magnetic nanoparticles of step 3), mix, polypeptide be coupled to magnetic nanoparticle surface, obtain final product.
Preferably, the solution of magnetic nanoparticles that polypeptide solution and the streptavidin of described biotin modification are modified mixes with the mass ratio of 0.01:5 ~ 4:5, and preferred mass is than being 1:5 ~ 3:5, and this mass ratio can obtain better effect.
Preferably, in 4 ~ 37 DEG C of mixing 0.5 ~ 2h.
Preferably, in 20 ~ 30 DEG C of mixing 10min ~ 2h.
Preferably, mixing 30 ~ 40min, this time, the used time was less under guaranteeing to obtain good effect.
Preferably, mix on constant-temperature table.
More preferably, mix on the constant-temperature table of 4 DEG C.
Also preferably, the rotating speed of shaking table is 150rpm.
Again on the one hand, the invention provides the polypeptide-magnetic nanoparticle for specific recognition tumour cell described in the polypeptide for specific recognition tumour cell described in a kind of the invention described above or the invention described above for the preparation of enrichment, separation or the application that detects in the product of circulating tumor cell.
Preferably, described product is test kit or reagent.
Another aspect, the invention provides the polypeptide for specific recognition tumour cell described in a kind of the invention described above or the application of polypeptide-magnetic nanoparticle in the medicine for the preparation for the treatment of metastases relative disease for specific recognition tumour cell described in the invention described above.
Preferably, described metastases relative disease is the tumour that cell epithelia adhesion factor (EpCAM) high expression level is relevant.
More preferably, the tumour that described cell epithelia adhesion factor high expression level is relevant comprises prostate cancer, mammary cancer and lung cancer.
Also on the one hand, the invention provides the method for one peptide species-magnetic nanoparticle concentration and separation circulating tumor cell, comprise the following steps:
Steps A: the polypeptide solution preparing biotin modification, prepares the solution of magnetic nanoparticle dispersion, and the mixing solutions of biotin modification polypeptide and magnetic nanoparticle, mixes in shaking table.
Step B: by the solution in mixed steps A, after collecting by ferromagnetism attraction, sucking-off solution, then uses the PBS buffered soln of pH=7.2 by resuspended for the particle of magnet enrichment, then uses magnet enrichment, 3 times so repeatedly, resuspended stand-by with the PBS of pH=7.2.
Step C: 4 volume % paraformaldehydes are fixed, the people of breast cancer cell (MCF-7) and the EpCAM feminine gender of the EpCAM positive of Hoechst.33342 dyeing early young grain acute leukemia cells (HL-60) with 50:2E7, the ratio mixing of 50:2E8,50:2E9 is mixed into the PBS solution of 1ml respectively.
Step D: the magnetic nanoparticle after modifying in the step B that takes a morsel adds cell mixture in step C or blood sample to be measured, mixes on shaking table.
Step e: take off the mixed solution in step D, collects magnetic nanoparticle by ferromagnetism attraction, discards enchylema, with the magnetic nanoparticle of the resuspended enrichment of PBS, then use magnet enrichment, 3 times so repeatedly.
Step F: add up isolated cell and take pictures under microscope.
Preferably, described steps A specifically comprises the following steps:
Steps A .1: be dissolved in by polypeptide in damping fluid, preferably, is dissolved into polypeptide in the phosphate buffered saline buffer of pH=6.8 ~ 7.5, more preferably, is dissolved into by polypeptide in the PBS solution of pH=7.2;
Steps A .2: extracted by the magnetic nanoparticle be scattered in water, is moltenly dispersed in a small amount of pH=6.8 ~ 7.5 phosphate buffered saline buffer, preferably, is dissolved in the PBS solution of pH=7.2 by magnetic nanoparticle polypeptide, and on vortex instrument vortex 3 ~ 5min;
Steps A .3: polypeptide solution obtained in is A.1 joined obtained blended liquid in the magnetic nanoparticle A.2, preferably, the mass ratio of polypeptide and magnetic nanoparticle is 1:5 ~ 3:5, preferably, this mixed solution, in 25 DEG C, the constant-temperature table of 150rpm mixes 30 ~ 40min;
Preferably, in step B and step e, during rich magnetic nano particle, use ferromagnetism magnet, preferably, in particle concentrations process, constantly rock by enrichment solution, more preferably, magnet enrichment process continues at least 10min/ time;
Preferably, described step C specifically comprises the following steps:
Step is C.1: get a certain amount of MCF-7, with fixed cell 20 ~ 40min under the paraformaldehyde room temperature of 4 volume %, preferably, fix 30min under room temperature.
Step is C.2: utilize PBS damping fluid to remove and wash away cell stationary liquid, under room temperature effect, cell dyeing 30 ~ 50min is carried out with the Hoechest.33342 of 60 ~ 120 μ g/ml, preferably, the Hoechest.33342 of 80 μ g/ml effects at room temperature carries out cell dyeing 30min.
Preferably, in step D, the amount of the magnetic nanoparticle added is 5 ~ 10 μ l, more preferably, adds 5 μ l.
Concrete in order to obtain technological method of the present invention, invention has been the test and study of the following aspects:
1) a series of fluorescein isothiocyanate (FITC has been designed and synthesized, fluoresceinisothiocyanate)-polypeptide-vitamin H (Biotin), with the HL-60 cell of MCF-7 and the EpCAM feminine gender of the EpCAM positive for tested object, pass through Flow Cytometry, find that Pep7-2 and Pep7-3 of design can specific binding EpCAM positive cell, EpCAM negative cells combination rate is low, and binding constant is proper; And for negative cells, Pep7 in contrast shows certain negative binding characteristic, but negative combination rate is higher, and selectivity is lower;
Described process of the test comprises the steps:
A1: the polypeptide of design is made solution;
B1: get the solution that a certain amount of step a1 obtains, blended with MCF-7 and HL-60 cell, hatch 30min for 4 DEG C.
C1: make certain density (>=10 after utilizing PBS damping fluid to wash away unconjugated polypeptide
6individual/ml) cell suspension.
Suspension is detected on flow cytometer polypeptide and cell in conjunction with situation.
2) by 1) in prove that the polypeptide solution of specific recognition EpCAM positive cell joins in magnetic nanoparticle, be prepared into polypeptide-magnetic nanoparticle;
The preparation method of the polypeptide-magnetic nanoparticle of described specific recognition EpCAM comprises the steps:
A2: fully mixed with magnetic nanoparticle by described FITC-polypeptide-Biotin, forms mixed solution;
B2: and the solution in above-mentioned a2 is mixed 30 ~ 40min with the rotating speed of 150rpm on 25 DEG C of shaking tables.
C3: remove unconjugated polypeptide solution, is resuspended in polypeptide-magnetic nanoparticle in PBS.
Fluorescent microscope and the polypeptide-magnetic nanoparticle of laser particle analyzer to synthesis is utilized to characterize.
Preparing in polypeptide-magnetic nanoparticle, adopting constant-temperature table fully to mix.Remove unconjugated polypeptide solution, preferably use strong magnets, preferred adsorption time is 10min, in order to prevent magnetic nanoparticle to run off, to discarding solution, with suction pipe from the opposite side of particle aggregation by its slow sucking-off.
In addition, before preparing polypeptide-magnetic nanoparticle, also comprise the step of dispersed magnetic nano particle, the method dispersed magnetic nano particle of ultrasonic/vortex can be adopted.
3) fixed by the paraformaldehyde of cell with 4 volume %, the breast cancer cell (MCF-7) after Hoechst.33342 dyeing and people early young grain acute leukemia cells (HL-60) mix with the ratio of 50:2E7,2E8,2E9, are dissolved in the PBS solution of 1ml respectively.
A3: get a certain amount of MCF-7 cell, with fixed cell under the paraformaldehyde room temperature of 4 volume % 30 minutes.
B3:PBS removed cell stationary liquid, by 80 μ g/mlHoechest.33342 room temperature function cells 30 minutes.
C3: after cell counting count board counts two kinds of cells, is settled to 995 μ l after getting appropriate mixing.
4) polypeptide-magnetic nanoparticle that takes a morsel adds cell mixture in step 3) or blood sample to be measured, mixes, utilize magnet that MCF-7 cell is carried out to enrichment and is separated on shaking table.
Particularly, enrichment is as follows with the step being separated MCF-7 cell:
A4: get 5 μ l polypeptide-magnetic nanoparticles, join in the sample to be separated of 995 μ l.
B4: polypeptide-magnetic nanoparticle is placed on the constant-temperature table of 150rpm in the mixed solution of cell sample, jointly hatches 30min at 4 DEG C.
C4: ferromagnetism magnet adsorption magnetic nanoparticle 10min, and constantly rock mixed solution, from the opposite side of enrichment positions, by unconjugated cell suspension sucking-off, then use the resuspended magnetic nanoparticle of PBS, then rich magnetic nano particle, 3 times so repeatedly.
5) under microscope, cell count is added up.
Concrete statistics and imaging comprise the steps:
A5: by the suspension of magnetic nanoparticle PBS damping fluid resuspended one-tenth 10 ~ 20 μ l collected above, in order to strengthen dispersiveness, can on vortex instrument vortex 3 ~ 5min.
B5: draw a certain amount of above-mentioned a6 suspension on slide glass, then cover with cover glass, i.e. available fluorescence microscope.
C5: under the microscope, gets the different visual field (at least 3) and adds up the cell of blue light-emitting in all cells in light field and details in a play not acted out on stage, but told through dialogues, take pictures.
In sum, the object of the invention is to develop the novel method of a kind of separation, enrichment and detection CTC, devise the polypeptide fragment that two have specific recognition CTC, be expected to alternative existing antibody, and in conjunction with the simple and quick enrichment of magnetic nanoparticle technology and separation of C TC, thus reduction Clinical CT C detects high cost greatly, and new thinking can be provided for Ag-Ab immune Research.
One embodiment of the invention: at cell levels, polypeptide can specific binding EpCAM positive cell to utilize flow cytometer to verify; The situation and the combination utilizing surface plasmon resonance technology (SurfacePlasmonResonancetechnology, SPR) to demonstrate this polypeptide fragment and anti-EpCAM antibody and EpCAM further is on a molecular scale dissociated.Utilize dynamic light scattering (DynamicLightScattering, DLS) and fluorescence technique characterize utilizing streptavidin to react obtained polypeptide-magnetic nanoparticle, particularly, record the zeta current potential of magnetic nanoparticle before and after coupling and the change of particle diameter by DLS, utilize fluorescent microscope to take pictures and show that the change to modifying front and back magnetic nanoparticle is observed.Utilize magnet to the gathering of magnetic nanoparticle, attraction, collecting action, CTC is carried out enrichment and is separated.Specifically comprise the steps:
Step 1: design and synthesize a series of FITC-polypeptide-Biotin, utilize the HL-60 cell of MCF-7 and the EpCAM feminine gender of Flow Cytometry and the EpCAM positive, Pep7-2, Pep7-3 and Pep7 of being surprised to find design can specific binding EpCAM positive cells, but for EpCAM negative cells, Pep7 shows certain negative binding characteristic, and selectivity is lower; And the combination rate of Pep7-2, Pep7-3 and EpCAM negative cells HL-60 is very low, selectivity is high;
Step 2: the combination-dissociation constant utilizing polypeptide fragment and anti-EpCAM antibody and the EpCAM in SPR technique detecting step 1 with specific recognition CTC, the avidity that result is surprised to find Pep7-2, Pep7-3 and target protein EpCAM is suitable with antibody, and the avidity of Pep7 and EpCAM positive cell is slightly low;
Step 3: specific recognition polypeptide step 2 proved carries out biotin modification, and the magnetic nanoparticle mixing 30min its solution and streptavidin modified, be prepared into polypeptide-magnetic nanoparticle;
Step 4: utilize DLS, fluorescence microscopy and MALDI-TOF analytical reagent composition to characterize the polypeptide-magnetic nanoparticle in step 3;
Step 5: fix utilizing the paraformaldehyde of 4 volume %, and after utilizing Hoechst.33342 to dye MCF-7 Breast Cancer Cell and people morning young grain acute leukemia cells HL-60 cell with 50:2E7, the ratio of 50:2E8,50:2E9 is mixed into the PBS solution of 1ml respectively; Wherein 2E7,2E8,2E9 represent that negative cells number is 2 × 10 respectively
7individual, 2 × 10
8individual, and 2 × 10
9individual.
Step 6: the polypeptide-magnetic nanoparticle that takes a morsel adds cell mixture in step 3 or blood sample to be measured, mixes 30min, enrichment and separation of C TC on shaking table.
Step 7: add up light and shade cell after the match and take pictures under microscope.
The present invention devises a series of polypeptide fragment, and has found that two have the specific polypeptide fragment of specific recognition EpCAM; The combination utilizing SPR to analyze these two polypeptide fragments and anti-EpCAM antibody and EpCAM further is on a molecular scale dissociated situation characteristic.
The present invention utilizes the strong interaction of biotin-streptavidin, by the magnetic nanoparticle coupling that the specific recognition polypeptide of biotin modification and streptavidin are modified, DLS is utilized to characterize the change of magnetic nanoparticle Zeta potential before and after coupling, the fluorescigenic magnetic nanoparticle after modification FITC-polypeptide-Biotin with fluorescence microscope; And prove that polypeptide-magnetic nanoparticle of the present invention can be used in the separation of CTC, enrichment and detection by test, and two peptide species of the present invention modify after nano particle separation rate of CTCs in the CTCs sample to simulation reach more than 80%, circulation ratio is fine, and its research has important meaning to the diagnosis of clinical tumor transfer and the prognosis of tumour.
The present invention proposes a kind of polypeptide that utilizes and the novel method of specific recognition is carried out to cell-surface antigens, and invented the polypeptide-magnetic nanoparticle material of a kind of new enrichment, separation and detection CTC, provide a kind of enrichment, separation and detection CTC quick, accurately and the detection technique of low cost, for the diagnosis of clinical tumor transfer patient, Treatment and Prognosis provide effective ways.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 shows the fluorescence photo after specific recognition polypeptide (volumetric molar concentration is 50 μMs) and MCF-7 cytosis; A, B, C and D are the fluorescence photo after PBS, 50 μMs of Pep7,50 μMs Pep7-2 and 50 μM Pep7-3 effect MCF-7 cell 30min successively;
Fig. 2 shows the fluorescent microscopy images of Pep7-2-magnetic nanoparticle and Pep7-3-magnetic nanoparticle; Wherein A, B represent respectively Pep7-2-magnetic nanoparticle bright field and blue-light excited under photo; C, D represent respectively Pep7-3-magnetic nanoparticle bright field and blue-light excited under photo;
Fig. 3 shows that the absorbancy of Pep7-2 and Pep7-3 at 495nm place is with the relation of change in concentration and fitting a straight line, and wherein, wherein Fig. 3 a represents the concentration-OD curve of Pep7-2, and Fig. 3 b represents the concentration-OD curve of Pep7-3;
Fig. 4 shows that Pep7-2-magnetic nanoparticle and Pep7-3-magnetic nanoparticle are to MCF-7 cell enrichment and the fluorescent microscopy images be separated; Wherein, A, B represent respectively Pep7-2-magnetic nanoparticle be separated CTC bright field and purple light excited under photo; C, D represent the photo of CTC under bright field and purple light optical excitation that Pep7-3-magnetic nanoparticle is separated respectively; E, F represent respectively magnetic nanoparticle be separated CTC bright field and purple light excited under photo;
Fig. 5 shows Pep7-2-magnetic nanoparticle and the enrichment of Pep7-3-magnetic nanoparticle to MCF-7 cell and the statistics of separation efficiency, in figure, MINPs-Pep7-3 represents Pep7-3-magnetic nanoparticle, MINPs-Pep7-3 represents Pep7-2-magnetic nanoparticle, 2E7 represents that MCF-7 cell and HL-60 cell count ratio are 50:2E7,2E8 represents that MCF-7 cell and HL-60 cell count ratio are that 50:2E8,2E9 represent that MCF-7 cell and HL-60 cell count ratio are 50:2E9.
Embodiment
Unless specifically stated otherwise, breast cancer cell (MCF-7) used in following examples, people early young grain acute leukemia cells (HL-60) all purchased from Academy of Medical Sciences basic scientific research cell bank.
Unless specifically stated otherwise, reagent used in following examples is analytical reagent, and can be commercially available from regular channel.
the checking of embodiment 1. polypeptid specificity identification EpCAM
The sequence of the polypeptide 1, used, reagent and checking cell
Polypeptide fragment (synthesized by Shanghai Ke Tai Bioisystech Co., Ltd, purity is 98 quality %), as shown in table 1 below.Magnetic nanoparticle: streptavidin-magnetic nanoparticle (solulink, ST022912, the U.S.).
Table 1
Using MCF-7 cell consistent for known report as EpCAM positive cell, HL-60 cell verifies the polypeptide fragment of specific recognition EpCAM as EpCAM negative cells.
2, concrete grammar
1) checking of the specific polypeptide of the EpCAM positive.
First the PBS of a series of FITC-polypeptide pH=7.2 designed and synthesized is made into the mother liquor of 1mg/ml; Collect a certain amount of (>=10
6individual) MCF-7 and HL-60 cell in the centrifuge tube of 1.5 μ l, add each polypeptide mother liquor of 7 μ l, and be diluted to 125 μ g/ml with PBS; Blow even with suction pipe by this mixed solution, 4 DEG C of mixing 30min are then the whizzer (LD5-2A of 1000rpm at rotating speed, Beijing Lei Boer whizzer company limited) in centrifugal 3min, supernatant discarded, then use PBS resuspended, so the method for 3 times removes unconjugated polypeptide repeatedly, finally by the solution of resuspended for cell one-tenth 1ml, with flow cytometer (FCM, BDFACSAria, BD, the U.S.) detect, statistic mixed-state result, as described in Table 2.
Table 2
As shown in table 2, the detected result of composite antibody, known, MCF-7 is EpCAM positive cell, and HL-60 is EpCAM negative cells, with have been reported consistent.
Use Pep7, Pep7-2 and Pep7-3 of homogenous quantities volumetric concentration again, then by and above-mentioned 1) act on the identical cell of equivalent and disposal methods under identical condition.For positive cell, Pep7, Pep7-2 and Pep7-3 all show good recognition capability (combination rate is respectively 91.71%, 87.85% and 86.67%), substantially suitable compared with the positive combination rate of antibody (92.76%); For negative cells, Pep7 shows certain negative binding characteristic (55.94%), but negative combination rate is higher, Pep7-2 and Pep7-3 all shows obviously low negative combination rate (9.22% and 9.45%), substantially suitable with the negative combination rate (2.20%) of antibody.Comprehensive above flow cytometry results, shows that Pep7-2 and Pep7-3 shows Selective recognition to EpCAM and combination.
MCF-7 cell is inoculated in the 96 flat blackboards in hole with the density in 5000/hole, spending the night, it is adherent to treat, the FITC-polypeptide (Pep7, Pep7-2 and Pep7-3) of 50 μMs is added in different hole respectively, 4 DEG C of effect 30min, then wash away unconjugated polypeptide gently with PBS, totally 3 times, add 100 μ l/ hole PBS every hole in the 96 flat blackboards in hole and add the PBS damping fluid of 100 μ l, and by PBS solution as negative control, at fluorescence inverted microscope (IX71, the OLYMPUS of 20 × camera lens, Japan), blue-light excited, take pictures under 20 × camera lens, the results are shown in Figure 1.
By Fig. 1, we are known, with the cell of the polypeptide effect equivalent of volumetric molar concentration, under identical treatment condition, the fluorescence intensity of homopolypeptide is not different, the fluorescence of specific recognition polypeptide Pep7, Pep7-2 and Pep7-3 is all comparatively strong, and can verify streaming result above, the bonding force of these three polypeptide fragments to EpCAM positive cell MCF-7 is stronger.
Ultrapure water (MilliQ) is used to be configured to the solution that concentration is 5mg/ml, 5mg/ml, 5mg/ml and 12.5ug/ml respectively Pep7, Pep7-2, Pep7-3 and anti-EpCAM antibody (purchased from Biolegend company), get 5 μ l and dropped in carboxylated SPR chip (purchased from Plexera company, the substrate of KxV5 type SPR standard configuration) on, by amino acid condensation reaction, polypeptide is fixed on SPR chip.Be then that EpCAM albumen (purchased from the R & D company) aqueous solution of 1.563 μ g/mL, 3.125 μ g/mL, 6.25 μ g/ml, 12.5 μ g/ml and 25 μ g/ml is as flow combinations phase by concentration series, the phosphoric acid of 1/20 is elutriant, with the flow velocity of 2 μ l/s in conjunction with 240s, dissociate 200s, carry out SPR analysis, and calculations incorporated-dissociation constant, result is as described in Table 3;
Table 3
From table 3, we can find out that polypeptide Pep7-2 and Pep7-3 and EpCAM has stronger combination, with the binding constant 3.72 × 10 of antibody
9compare, the binding constant of Pep7-2, Pep7-3 is respectively 3.39 × 10
8, 5.06 × 10
8close with it, and the binding constant of Pep7 and EpCAM is 6.62 × 10
7, avidity is more weak a little, and thus, the avidity that specific recognition polypeptide Pep7-2 and Pep7-3 is strong to target protein is its specific recognition and provides foundation as the surrogate of antibody.
specific recognition polypeptide Pep7-2 and Pep7-3 is passed through biotin-streptavidin by embodiment 2.
interact and magnetic nanoparticle coupling
1. get the magnetic nanoparticle 250 μ l of 5mg/ml, in vortexmixer vortex instrument (WH-866, Taicang Hua Lida experimental installation company limited) upper mixing 3 ~ 5min, collection is gone out with ferromagnetism magnet, and remove solvent, then get each 500 μ l of PBS solution of the FITC-Pep7-2-vitamin H (Biotin) of 1mg/ml and the FITC-Pep7-3-vitamin H (Biotin) of 1mg/ml, mix with magnetic nanoparticle respectively (mass ratio of polypeptide and magnetic nanoparticle is 2:5), then be placed on shaking table (THZ-D, Taizhou City Wanda air spring company limited) at 25 DEG C, 40min is mixed under 150rpm.Then use ferromagnetism magnet rich magnetic nano particle in centrifuge tube side, from the opposite side imbitition of centrifuge tube after about 10min, add a certain amount of PBS damping fluid again, resuspended magnetic nanoparticle, then enrichment, suck liquid, so 3 times repeatedly, unconjugated polypeptide can be removed, obtain FITC-Pep7-2-vitamin H (Biotin)-magnetic nanoparticle and FITC-Pep7-3-vitamin H (Biotin)-magnetic nanoparticle, finally use the PBS buffer solution of 250 μ l respectively again.
2. in addition, get the magnetic nanoparticle 250 μ l of 5mg/ml, 3 ~ 5min is mixed on vortexmixer vortex instrument, collection is gone out with ferromagnetism magnet, and remove solvent, then get each 2.5 μ l of PBS solution of 1mg/mlFITC-Pep7-2-vitamin H (Biotin) and 1mg/mlFITC-Pep7-3-vitamin H (Biotin), 250 μ l, 750 μ l, 1ml mixes with magnetic nanoparticle respectively, and (mass ratio of polypeptide and magnetic nanoparticle is respectively 0.01:5, 1:5, 3:5, 4:5), then be placed on shaking table at 25 DEG C, under 150rpm, mix 10min respectively, 30min, 1h and 2h.Then use ferromagnetism magnet rich magnetic nano particle in centrifuge tube side, from the opposite side imbitition of centrifuge tube after about 10min, add a certain amount of PBS damping fluid again, resuspended magnetic nanoparticle, then enrichment, suck liquid, so 3 times repeatedly, unconjugated polypeptide can be removed, obtain FITC-Pep7-2-vitamin H (Biotin)-magnetic nanoparticle and FITC-Pep7-3-vitamin H (Biotin)-magnetic nanoparticle, finally use the PBS buffer solution of 250 μ l respectively again.
the sign of embodiment 3. pairs of polypeptide-magnetic nanoparticles
In Example 21 FITC-Pep7-2-vitamin H (the Biotin)-magnetic nanoparticle obtained and FITC-Pep7-3-vitamin H (Biotin)-solution of magnetic nanoparticles (mass ratio of polypeptide and magnetic nanoparticle is 2:5) are a little, take pictures under microscope, result as shown in Figure 2, A and B be Pep7-2-magnetic nanoparticle respectively light field and blue-light excited under photo, the magnetic nanoparticle modifying Pep7-2 under blue light, sends green glow (absorption of FITC and emission wavelength are 495nm and 535nm); C and D be Pep7-3-magnetic nanoparticle respectively light field and blue-light excited under photo, modify pep7-3 magnetic nanoparticle under blue light, send green glow; Show polypeptide and magnetic nanoparticle coupling success.
FITC-Pep7-2-vitamin H (Biotin) and FITC-Pep7-2-vitamin H (Biotin) is used to configure 1 μ g/ml respectively, 50 μ g/ml, 100 μ g/ml, 200 μ g/ml, the PBS solution of 400 μ g/ml and 500 μ g/ml concentration is in photometer (TecaninfiniteM200, TECAN, SwissConfederation) absorbance (OD) of upper test polypeptide at 495nm place, the solution that Simultaneously test washes away in upper step is in the OD value at 495nm place, and typical curve and the equation (Fig. 3) of OD-concentration is simulated with origin software (OriginPro8.0 version), available formula (Eq. (1)) calculates the combination rate of magnetic nanoparticle and polypeptide,
The polypeptide amount that the polypeptide Liang – of the polypeptide amount of magnetic nanoparticle surface bonding=add elutes
Y=A+B*X(standard straight-line equation)
X=(Y-A)/B（1）
Y is the OD of polypeptide
495nm; A, B are the intercept that simulates of linear equation and slope respectively; X is by the OD recorded by linear equation
495nmthe amount of the polypeptide that value calculates.
The standard equation of composition graphs 3 and Eq. (1) can calculate Pep7-2, Pep7-3 and be respectively 21.2nmol/mg, 10.16nmol/mg in conjunction with the efficiency of magnetic nanoparticle.
The Pep7-2-magnetic nanoparticle taken a morsel and Pep7-3-magnetic nanoparticle are diluted to 1ml dispersion liquid respectively, transferred in zeta current potential pond, with laser particle analyzer (ZetasizerNanoZS, Malvern company limited, Britain) Zeta potential of analytic sample and median size, result is as described in Table 4.
Table 4
As shown in Table 4, after Pep7-2-FITC and Pep7-3-FITC modifies, the zeta current potential of magnetic nanoparticle becomes-36.4mV and-32.0mV from 42.5mV, particle diameter also slightly increases, the change of current potential is relevant with negative charge with polypeptide, and this result also illustrate that polypeptide is successfully coupled at (people such as D.-B.Shieh, Biomaterials2005 on magnetic nanoparticle, 26,7183 – 7191).
Further, get the Pep7-2-magnetic nanoparticle after obtained purifying and Pep7-3-magnetic nanoparticle, join respectively not containing in the pure water of ion, 60 DEG C of constant temperature dissociate 5 minutes, get dissociation solution supernatant and carry out MALDI-TOF analytical reagent composition (Karas, M.; Bahr, U. " LaserDesorptionIonizationMassSpectrometryofLargeBiomolec ules " .TrendsAnal.Chem.1990,9 (10): 321-5), result shows the characteristic peak having Pep7-2 and Pep7-3, thus proves that polypeptide has been coupled on magnetic nanoparticle really further.
In addition, (mass ratio of polypeptide and magnetic nanoparticle is respectively 0.01:5 in Example 22 other FITC-Pep7-2-vitamin H (Biotin)-magnetic nanoparticles obtained and FITC-Pep7-3-vitamin H (Biotin)-solution of magnetic nanoparticles, 1:5,3:5,4:5) carry out the characterization experiments identical with aforesaid operations step, above-mentioned similar result can be obtained.
embodiment 4. utilizes polypeptide-magnetic nanoparticle to carry out the separation of CTC, enrichment and detection
Collect a certain amount of MCF-7 cell, then fix 30min by the paraformaldehyde room temperature of 4 volume %, stationary liquid is washed away afterwards with PBS, adding 20 μ l(again can be any one value in 20 ~ 50 μ l) the Hoechst.33342(available from Sigma of 80 μ g/ml) dye (see people such as Q.Yuan to cell, Biochem.Biophys.Res.Commun.2007,356,880885), room temperature 30min; With cell counting count board to cell counting.Collect HL-60 cell, and count, then get appropriate MCF-7 cell and HL-60 cell, with MCF-7 cell and HL-60 cell count ratio for 50:2E7,50:2E8 and 50:2E9 is mixed and made into the enchylema that volume is 995 μ l, then add the polypeptide-solution of magnetic nanoparticles of 5 μ l above, the shaking table of 150rpm mixes 30min at 4 DEG C.Then, with ferromagnetism magnet rich magnetic nano particle in centrifuge tube side, from the opposite side imbitition of centrifuge tube after about 10min, add a certain amount of PBS again, resuspended magnetic nanoparticle, then enrichment, removing liquid, 3 times so repeatedly, obtains the MCF-7 cell of separation.Used the PBS of 20 μ l resuspended, get 10 μ l and drop on slide glass, after cover glass covers, make sample, statistical counting under fluorescent microscope again, the results are shown in Figure 4 and Fig. 5.
Fig. 4 A with 4B is that Pep7-2-magnetic nanoparticle carries out enrichment and the experimental result that be separated with HL-60 cell quantity than the mixing sample for 50:2E9 to the MCF-7 cell of 1ml; Fig. 4 C with 4D is that Pep7-3-magnetic nanoparticle carries out enrichment and the experimental result that be separated with HL-60 cell quantity than the mixing sample for 50:2E9 to the MCF-7 cell of 1ml; Fig. 4 E with 4F is that magnetic nanoparticle carries out enrichment and the experimental result that be separated with HL-60 cell quantity than the mixing sample for 50:2E9 to the MCF-7 cell of 1ml, contrast light field (4A, 4C, 4E) and the photo under details in a play not acted out on stage, but told through dialogues (4B, 4D, 4F), we can see, the magnetic nanoparticle after peptide modified can from 1ml up to 2 × 10
9a small amount of EpCAM positive cell MCF-7 is isolated, this cell proportion and clinical middle CTC content (0 ~ 50CTCs/10 in blood in the cell mixing of individual EpCAM negative cells
10ml hemocyte) close, therefore separating resulting has clinical in meaning.
As shown in Figure 5, further statistics shows, the separation rate of the nano particle CTCs in the CTCs sample to simulation after the modification of this two peptide species reaches more than 80%, circulation ratio is fine, therefore the prognosis of the diagnosis shifted clinical tumor of the research of Pep7-2-magnetic nanoparticle and Pep7-3-magnetic nanoparticle and tumour has important meaning.
Claims (23)
1., for a polypeptide for specific recognition circulating tumor cell, the aminoacid sequence of described polypeptide is the aminoacid sequence shown in following SEQIDNO:1:
VRRDAPRFSMQGLDACGGNX
1X
2(X
3)
n1(X
4)
n2
Wherein, X
1for Cys or Asn;
X
2for Cys or Asn;
X
3for Asn, n1=0 or 1;
X
4for Asn, n2=0 or 1.
2. the polypeptide for specific recognition circulating tumor cell according to claim 1, is characterized in that, the aminoacid sequence of described polypeptide is selected from the aminoacid sequence shown in SEQIDNO:2 and SEQIDNO:3.
3. for polypeptide-magnetic nanoparticle of specific recognition circulating tumor cell, its contain magnetic nanoparticle and with the polypeptide for specific recognition circulating tumor cell described in the claim 1 or 2 of its phase coupling.
4. polypeptide-the magnetic nanoparticle for specific recognition circulating tumor cell according to claim 3, is characterized in that, the mass ratio of described polypeptide and magnetic nanoparticle is 0.01:5 ~ 4:5.
5. polypeptide-the magnetic nanoparticle for specific recognition circulating tumor cell according to claim 4, is characterized in that, the mass ratio of described polypeptide and magnetic nanoparticle is 1:5 ~ 3:5.
6. polypeptide-the magnetic nanoparticle for specific recognition circulating tumor cell according to any one of claim 3 to 5, is characterized in that, described magnetic nanoparticle is the magnetic nanoparticle that streptavidin is modified.
7. polypeptide-the magnetic nanoparticle for specific recognition circulating tumor cell according to claim 6, is characterized in that, the particle diameter of described magnetic nanoparticle is 600 ~ 1500nm.
8. polypeptide-the magnetic nanoparticle for specific recognition circulating tumor cell according to claim 7, is characterized in that, the particle diameter of described magnetic nanoparticle is 800nm.
9. the preparation method of the polypeptide-magnetic nanoparticle for specific recognition circulating tumor cell according to any one of claim 3 to 8, described method comprises the steps:
1) polypeptide for specific recognition circulating tumor cell described in claim 1 or 2 is prepared;
2) by step 1) in polypeptide make the polypeptide solution of biotin modification;
3) magnetic nanoparticle that streptavidin is modified is dissolved in damping fluid and makes solution;
4) by step 2) in polypeptide solution and the step 3 of biotin modification) solution of magnetic nanoparticles mix after, mix, polypeptide be coupled to magnetic nanoparticle surface, obtain final product.
10. the preparation method of the polypeptide-magnetic nanoparticle for specific recognition circulating tumor cell according to claim 9, wherein, step 3) also comprise the step of being undertaken by method that is ultrasonic or vortex by solution of magnetic nanoparticles disperseing.
11. the preparation method of the polypeptide-magnetic nanoparticle for specific recognition circulating tumor cell according to claim 9, the solution of magnetic nanoparticles that polypeptide solution and the streptavidin of described biotin modification are modified mixes with the mass ratio of 0.01:5 ~ 4:5.
The preparation method of the 12. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 11, wherein, the mixing quality of solution of magnetic nanoparticles modified with streptavidin of the polypeptide solution of described biotin modification is than being 1:5 ~ 3:5.
The preparation method of the 13. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 11, wherein, the polypeptide solution of described biotin modification mixes 0.5 ~ 2h with the solution of magnetic nanoparticles that streptavidin is modified in 4 ~ 37 DEG C.
The preparation method of the 14. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 11, wherein, the polypeptide solution of described biotin modification mixes 10min ~ 2h with the solution of magnetic nanoparticles that streptavidin is modified in 20 ~ 30 DEG C and mixes.
The preparation method of the 15. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 11, mixing 30 ~ 40min.
The preparation method of the 16. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 15, wherein, mixes on constant-temperature table.
The preparation method of the 17. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 16, wherein, mixes on the constant-temperature table of 4 DEG C.
The preparation method of the 18. polypeptide-magnetic nanoparticles for specific recognition circulating tumor cell according to claim 17, wherein, the rotating speed of shaking table is 150rpm.
Polypeptide-the magnetic nanoparticle for specific recognition tumour cell according to any one of 19. polypeptide for specific recognition tumour cell according to claim 1 and 2 or claim 3 to 8 is for the preparation of enrichment, separation or the application that detects in the product of circulating tumor cell.
20. application according to claim 19, wherein, described product is test kit or reagent.
21. polypeptide for specific recognition tumour cell according to claim 1 and 2 or the application of polypeptide-magnetic nanoparticle in the medicine for the preparation for the treatment of metastases relative disease for specific recognition tumour cell according to any one of claim 3 to 8.
22. application according to claim 21, wherein, described metastases relative disease is the tumour that cell epithelia adhesion factor high expression level is relevant.
23. application according to claim 22, wherein, the tumour that described cell epithelia adhesion factor high expression level is relevant comprises prostate cancer, mammary cancer and lung cancer.
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| CN104535772B (en) * | 2014-12-22 | 2016-11-23 | 国家纳米科学中心 | Polypeptide-magnetic nanoparticle of the circulating tumor cell of HER2 high expressed and its preparation method and application |
| CN105440112A (en) * | 2015-12-07 | 2016-03-30 | 国家纳米科学中心 | Polypeptide-albumin coupling drug and preparing method and application thereof |
| CN110501208B (en) * | 2018-05-17 | 2023-06-27 | 国家纳米科学中心 | Folic acid functionalized streptavidin modified magnetic nanoparticle, preparation method and application thereof |
| CN109112107A (en) * | 2018-09-11 | 2019-01-01 | 上海浦美医学科技有限公司 | A method of separation CTC is captured based on rgd peptide |
| CN111983220B (en) * | 2019-05-21 | 2024-03-19 | 北京中科纳泰生物科技有限公司 | Polypeptide magnetic nanoparticle, preparation method and application thereof |
| CN110579605A (en) * | 2019-08-13 | 2019-12-17 | 姜宇腾 | method for quickly separating immunomagnetic beads |
| WO2021102919A1 (en) * | 2019-11-29 | 2021-06-03 | 京东方科技集团股份有限公司 | Peptoid compound and detection chip coupled on surface thereof with peptoid compound |
| CN111632202A (en) * | 2020-06-29 | 2020-09-08 | 南开大学 | Tumor cell adhesion material and preparation method and application thereof |
| CN112410302B (en) * | 2020-11-10 | 2023-02-17 | 福建医科大学 | Multi-target combination based tumor stem cell enrichment and screening method |
| CN116884506B (en) * | 2023-09-06 | 2023-11-17 | 北京科技大学 | Method and system for predicting enrichment of drug-loaded nano-particles at different temperatures in vessel wall |
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