CN110412262A - It is a kind of identify lymphatic endothelial cells magnetic probe and its preparation - Google Patents
It is a kind of identify lymphatic endothelial cells magnetic probe and its preparation Download PDFInfo
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Abstract
The present invention provide it is a kind of identify lymphatic endothelial cells magnetic probe and its preparation.The magnetic probe includes: hud typed Fe3O4@KCTS magnetic nanoparticle and modification are in the hud typed Fe3O4The lymphatic endothelial cells double antibody on@KCTS magnetic nanoparticle surface；Wherein, the lymphatic endothelial cells double antibody is LYVE-1 antibody or its antibody fragment and Podoplanin antibody or its antibody fragment.Fe3O4@KCTS-Double antibody magnetic nano-particle is directly sorted with cell incubation, obtains high-purity lymphatic endothelial cells.Through comparing, Fe3O4The double dye rates of the cell that@KCTS-Double antibody is sorted reach 88.9%, and Fe3O4The cell that@KCTS-Double antibody is sorted is higher at pipe ability, and phagocytic activity is stronger, and bimodal can be imaged on diagnosing tumor.
Description
Technical field
The invention belongs to field of medicaments, and in particular to it is a kind of identify lymphatic endothelial cells magnetic probe and its preparation.
Background technique
More and more evidences show that tumor microenvironment influences oncobiology characteristic, especially tumour associated transitions.But
It is that many related potential mechanism are still very unclear, the lymphatic endothelial cells especially in lymphatic vessel.In order to study lymph
Effect of the endothelial cell in cancer Lymph Node Metastasis needs a kind of reliable model.Due to lacking the mark of lymphatic vessel specificity
Note is especially to discriminate between them from blood vessel endothelium, the function investigation seldom report of lymphatic endothelial cells.In recent years, it is determined that
Some Specific markers of lymphatic endothelial cells, such as lymphatic endothelial HA receptor 1 (LYVE-1), Podoplanin, blood vessel
Endothelial growth factor receptor 3 (VEGFR-3), Prox-1 and D2-40.Based on these progress, immunological magnetic bead sorting skill has been used
Art successfully enriches in vasculolymphatic normal tissue separation lymphatic endothelial cells (LEC) from having, but and molecular pathology
Used in many other markers it is the same, no one of LEC related molecular marker object is completely special to LEC.Due to prox-
1 transient expression in nucleus, so being difficult to use in clinical application.Although VEGFR-3 is expressed in LEC, it is in cancer
Middle shortage lymphatic vessel specificity, because it is also expressed in vascular endothelia.LYVE-1 is specific expressed in LECs, removes normal hepatocytes
Outside blood sinus, spleen endothelium, High endothelial venulae and activation tissue macrophages, it is not present in vascular endothelia.Podoplanin is
The Specific marker of LECs is not to be expressed in vascular endothelial.Therefore divided at present just for some target
From lymphatic endothelial cells, the lymphatic endothelial confluent monolayer cells purity obtained after sorting is not high.Moreover, because technological challenge, seldom
Report the sorting of tumour associated lymphatic endothelial cell.Magnetic ferroferric oxide, nanogold, quantum dot, liposome, titanium oxide etc.
Nano particle due to good biocompatibility, large specific surface area, be easy to modify the features such as other biological functional molecular, widely apply
In cancer diagnosis and treatment.Magnetic Fe3O4Nano particle in addition to have the characteristics that it is above other than, because it is with narrow size distribution, good
Good dispersed, high paramagnetism, controllable size and be widely used in Magnetic resonance imaging.In recent years, it is based on superparamagnetism
The knub thermotherapy of iron oxide emerges, magnetic control embolization technique, Magnetic Fluid Hyperthermia equipment, be coupled anticancer drug tumor target
Also there is in-depth study to medicine-feeding technology.Wherein, in target administration technical field, with the coated Superparamagnetic Iron Oxide of antibody,
In cell separation, identification, the fields such as early diagnosis of tumour are also widely used.
Since Magnetic particles partial size is small, surface can be high, therefore can reunite mutually.And when partial size is greater than 100nm, easily by blood vessel
Reticuloendothelial system is removed；But partial size is too small, and there is also the weak disadvantages of magnetic response.Therefore one layer of capsulating material of outsourcing is needed.
Chitosan has biocompatibility, blood compatibility as a kind of natural high molecular substance, surface hydroxyl rich in and amino
The good characteristics such as property, safety and microbic resolvability and receive significant attention.Currently, by chitosan or chitosan derivatives with
Magnetic Fe3O4In conjunction with can effectively prevent magnetic bead mutual after Magnetic particles have been coated with the polymer substances such as chitosan, antibody
Reunite, obtains the preferable magnetic fluid of dispersion performance.
At present still not over homemade dual-target magnetic bead from being isolated in mouse intestinal cancer in the higher lymphatic vessel of purity
Chrotoplast.
Summary of the invention
An object of the present invention is to provide a kind of magnetic probe.
Magnetic probe provided by the present invention, are as follows: the Fe of lymphatic endothelial cells (LEC) double antibody modification3O4@KCTS
(Fe3O4@KCTS-Double antibody) magnetic nano-particle, wherein KCTS is α-ketoglutaric acid contracting chitosan；
The magnetic probe includes: hud typed Fe3O4@KCTS magnetic nanoparticle and modification are described hud typed
Fe3O4The lymphatic endothelial cells double antibody on@KCTS magnetic nanoparticle surface；
Wherein, the lymphatic endothelial cells double antibody is LYVE-1 antibody or its antibody fragment and Podoplanin antibody
Or its antibody fragment.
The partial size of the magnetic probe can be 80-150nm, concretely 100nm, good dispersion, monodispersity index
It (PDI) can be 0.2-0.5, concretely 0.392, good biocompatibility.
Above-mentioned magnetic probe passes through the method included the following steps and is prepared:
1) chitosan is subjected to carboxylated modification with α-ketoglutaric acid, obtains the α-ketoglutaric acid that surface is rich in-COOH base
Contracting chitosan (KCTS)；
2) under the activation of carbodiimide, Fe3O4The covalent bond that magnetic nano-particle passes through the-COOH of surface-OH and KCTS
In conjunction with obtaining Fe3O4@KCTS magnetic nanoparticle；
3) two kinds of LEC antibody are covalently bonded to the Fe that step 2) obtains3O4@KCTS magnetic nanoparticle surface, obtains
Fe3O4@KCTS-Double antibody magnetic nano-particle, i.e. magnetic probe；
In step 3), described two LEC antibody specifics can be anti-for LYVE-1 antibody or its antibody fragment and Podoplanin
Body or its antibody fragment.
The concrete operations of above method step 2) are as follows: KCTS is dissolved in acetic acid solution, stirring is to being completely dissolved；It is added
The Fe of carbodiimide activation3O4Magnetic nano-particle, ultrasonic disperse；Reaction solution is gone into mechanical stirring, is added and contains span-80
Atoleine, be persistently stirred to react；Glutaraldehyde solution, mechanic whirl-nett reaction is added；After its completely reaction, solution is separated,
Collect precipitating, centrifuge washing；Finally obtained solid product is dried in vacuo, grinds, obtains Fe3O4@KCTS magnetic Nano
Grain；
KCTS and Fe3O4The mass ratio of magnetic nano-particle can be 1-5:1, concretely 1:1；
The concrete operations of step 3) are as follows: by Fe3O4@KCTS magnetic nanoparticle is dissolved in HEPES buffer solution, ultrasound point
It dissipates, EDC/NHS crosslinking agent is added, be incubated for, after having reacted, magnetic separation goes out the Fe of crosslinking activation3O4@KCTS；By two kinds of LEC
Antibody (LYVE-1 antibody or antibody fragment and Podoplanin antibody or antibody fragment) is added separately to above-mentioned crosslinking activation
Fe3O4It in@KCTS, mixes, is incubated for；Add bovine serum albumin closing；Magnetic separation obtains Fe3O4@KCTS-Double
Antibody magnetic nano-particle.
Wherein, Fe3O4The proportion of@KCTS magnetic nanoparticle and EDC/NHS crosslinking agent can be 1mg:100 μ L；
The pH of the HEPES buffer solution is 7.2；
The time of the ultrasonic disperse can be 30min；
It can be 30min that the time being incubated for after EDC/NHS crosslinking agent, which is added,；
LYVE-1 antibody or antibody fragment, Podoplanin antibody or antibody fragment and Fe3O4@KCTS magnetic nanoparticle
Proportion successively may be used are as follows: 1-10 μ g:1-10 μ g:1mg；
The LEC antibody is added in the form of PBS solution, and the concentration of antibody can be 10 μ g/mL in the solution.
The time being incubated for after antibody, which is added, to be 1-3h, concretely 2h；
The time that bovine serum albumin rear enclosed is added can be 0.5-2h, concretely 30min.
The Fe of above-mentioned magnetic probe, i.e. lymphatic endothelial cells double antibody modification3O4@KCTS(Fe3O4@KCTS- Double
Antibody) application of the magnetic nano-particle in lymphatic endothelial cells sorting also belongs to protection scope of the present invention.
It is a further object of the present invention to provide a kind of methods for sorting lymphatic endothelial cells.
The method of sorting lymphatic endothelial cells provided by the present invention, comprising:
By Fe3O4@KCTS-Double antibody magnetic nano-particle and mixing with cells to be sorted are incubated for, centrifugation, to receipts
Buffer is added in the solid precipitating collected, cell is resuspended, is eluted cell suction with magnet.
The temperature of the incubation can be 4-37 DEG C, and the time can be 15-60min.
The present invention is with magnetic Fe3O4Nano particle is core, poly- with α-ketoglutaric acid contracting shell by the activation of carbodiimide
Sugared (KCTS) crosslinking, is successfully prepared hud typed Fe3O4@KCTS magnetic nanoparticle is then repaired on magnetic nanoparticle surface
LYVE-1 and Podoplanin antibody is adornd, the magnetic double antibody nano-probe of LEC in specific recognition tumour is successfully constructed.
We remove tumor tissues from mouse intestinal cancer CT26 cell tumor model, shred completely and are divided into two parts.One
Part first contaminates antibody LYVE-1-PE using currently used, is then separated by Anti-PE MicroBeads；Another part makes
With homemade Fe3O4@KCTS-Double antibody magnetic nano-particle is directly sorted with cell incubation.Through comparing, Fe3O4@
The double dye rates of the cell that KCTS-Double antibody is sorted reach 88.9%, are much higher than the magnetic bead sorting of market purchase
Double dye rates 71.2%, and Fe3O4The cell that@KCTS-Double antibody is sorted is higher at pipe ability, phagocytic activity
It is stronger, and bimodal can be imaged on diagnosing tumor.This illustrates that we successfully develop one kind and are purified into from tumor tissues
The double antibody magnetic nano-probe of high-purity lymphatic endothelial cells is change of the lymphatic endothelial cells in intestinal cancer development process
Change and its provides the foundation in the research of clinical early stage bimodal imaging diagnosis.
Detailed description of the invention
Fig. 1 shows Fe3O4The characterization of@KCTS-LECs- double antibody magnetic nano-probe.A.Fe3O4@KCTS-LECs- is bis-
The transmission electron microscope image of antibody magnetic Nano probe (scale bar, 100nm).B. partial size, probe are detected by dimension analysis
Average-size be 91.28nm.C.Zeta current potential is about -32.8mv.D.Fe3O4@KCTS-LECs- double antibody nano-probe point
There is emission peak not under 488 and 545 exciting light.
Fig. 2 is vasculolymphatic expression in Colorectal Carcinoma.Lymphatic vessel in histotomy (is compared by immunohistochemistry
Example ruler, 100 μm) it is positive on lymphatic endothelial cells film to LYVE-1, the right is the picture of amplification.
Fig. 3 is lymphatic endothelial cells purity analysis.A. by LYVE-1 (APC) in Flow cytometry LEC and
Podoplanin (FITC) coexpression rate.B. flow cytometry statisticallys analyze, P < 0.05 *.C. pass through Calcein AM (green)
External test LEC pipe forms (scale bar, 50 μm).D. pass through LYVE-1 microballon and Fe3O4@KCTS-LECs- double antibody nanometer
The LEC of particle (scale bar, 100 μm) separation carries out endothelial cell phagocytosis measurement, and (DiI is marked, red；DAPI, it is blue
Color).
Specific embodiment
The present invention will be described below by way of specific embodiments, but the present invention is not limited thereto.
Experimental method used in following embodiments is conventional method unless otherwise specified；Institute in following embodiments
Reagent, biomaterial etc., are commercially available unless otherwise specified.
Embodiment
Material and method
Cell line and cell culture condition
In ATCC cell bank, CT26 cell line, which is used, contains 10% fetal calf serum for straight colon cancer cell line (CT26) purchase
The DMEM culture medium of (Fetal Bo vine Serum, FBS), 100U/mL penicillin and 100U/mL streptomysin are at 37 DEG C, 5%
CO2Cell incubator in cultivate.
Experimental animal model
The BALB/c mouse of 4-6 week old is got out, weight is between 150-200g；The CT26 for being collected in logarithmic phase growth is thin
Born of the same parents, PBS are washed 3 times, after being digested with pancreatin, are collected in centrifuge tube, and 1000rpm is centrifuged 5min；Cell is resuspended with PBS and adjusts
Whole cell concentration is 2 × 107/mL；Mouse armpit skin, every 200 μ of mouse subcutaneous injection cell suspension are wiped with cotton ball soaked in alcohol
L, routine observation tumour growth situation.Female/male BALB/c mouse of 4-6 week old is purchased from Guangxi Medical University's Experimental Animal Center,
Scheme used abides by Guangxi Medical University's animal protection and uses management rules, and rearing conditions reach SPF grades.
1, the Fe of LEC double antibody modification3O4@KCTS(Fe3O4@KCTS-Double antibody) magnetic nano-particle
Preparation
Chitosan (purchase is in the auspicious happiness biology in Xi'an) is first subjected to carboxylated modification with α-ketoglutaric acid, obtains surface richness
The α-ketoglutaric acid carboxymethyl chitosan (KCTS) of the base containing-COOH.Again with Fe3O4(purchase is in the biological Co., Ltd of the auspicious happiness in Xi'an)
For core, KCTS is basic skeleton structure, under the activation of carbodiimide, Fe3O4Nanoparticle by surface-OH and KCTS-
COOH synthesizes Fe by covalently bonded3O4@KCTS magnetic nano-composite particle.Method particularly includes: it weighs KCTS 0.4g and is dissolved in
In the acetic acid solution of 40mL 2%, to mechanical stirring to being completely dissolved；The carbodiimide activation that 0.4g is prepared then is added
Fe3O4Magnetic nano-particle, ultrasound 30min, makes it be uniformly dispersed under cell Ultrasonic Cell Disruptor；Reaction solution is gone into machinery
80mL atoleine (span-80 containing 1.5mL) is added in 250mL three-necked flask in stirring, and 500rpm is persistently stirred to react
2h；The glutaraldehyde solution of 2mL 2.5%, 500rpm mechanic whirl-nett reaction 2h is added；After its completely reaction, separated with magnetic frame
Solution collects precipitating, successively uses petroleum ether, acetone 6000rpm centrifuge washing 3 times；Finally obtained solid product is placed in very
For 24 hours, finally grinding obtains Fe for 70 DEG C of dryings in empty drying box3O4@KCTS；
By LEC antibody LYVE-1 and podoplanin by being covalently bonded to carboxylated under the activation of EDC/NHS
Fe3O4On@KCTS, the magnetic Nano probe (Fe of LEC double antibody modification is obtained3O4@KCTS-LEC- Doubleantibody),
It is coupled the Fe of LEC double antibody3O4Concentration used in@KCTS is 1mg/mL.Method particularly includes: by 1mg magnetic Fe3O4@KCTS is dissolved in
In 1mL HEPES buffer solution (pH=7.2), ultrasonic disperse 30min；100 μ L EDC/NHS crosslinking agents are added, is placed on shaking table and incubates
30min is educated, has been crosslinked Fe with magnetic frame separation after having reacted3O4@KCTS removes unreacted crosslinking agent；It is separately added into
The magnetic Fe that the antibody of the 10 μ g/mL concentration of 0.5mL was activated to above-mentioned crosslinking agent3O4In@KCTS, turbula shaker is mixed, permanent
2h is incubated under warm shaking table；Add the bovine serum albumin closing 30min of 100 μ L 1%；EP equipped with reaction mixture is managed
It is placed on magnetic frame, magnetic Fe3O4@KCTS is gathered in EP tube bottom in magnetic field, inhales after abandoning supernatant, gained magnetic Fe3O4@
KCTS particle is washed 3 times with 1mL coupling buffer again, and Magneto separate obtains the nano particle of double antibody coupling.It is kept in dark place in 4
℃。
2, the form of transmission electron microscope (TEM) observation nano particle
The Fe prepared is taken respectively3O4@KCTS-LEC-Double antibody is diluted；It is prepared above-mentioned
Solution be placed in sufficiently ultrasonic disperse 2h in ultrasonic wave dispersion instrument；Prepare copper mesh, a drop (10 μ L) is taken uniformly to spread on TEM copper mesh
On, several minutes of standing and drying at room temperature are completely dried to it.Copper mesh is put into TEM Electronic Speculum detection bar, Fe is observed3O4@KCTS-
The particle size and appearance form of LEC-Doubleantibody.
3, the particle size and current potential of dynamic laser partial size scatterometer detection nano material
Taking concentration is the Fe of 1mg/mL3O420 μ L of@KCTS-LEC-Double antibody nanoparticle is managed in 2mL EP
In, use ddH2O is diluted to 1mL；The above-mentioned solution prepared is placed in sufficiently ultrasonic disperse 2h in ultrasonic wave dispersion instrument；
Dynamic laser partial size scatterometer is opened, 10min is preheated；Take sample to be tested in clean quartz curette, the phase of selection detection sample
Parameter, the particle size (Size) of test sample, current potential (Zeta) are answered, while recording the dispersion index (PDI), every of each sample
A sample repeats detection three times.
4, immunohistochemistry
Tissue after being fixed with formalin carries out paraffin embedding.The Deparaffinized sections of CT26 transplantable tumor are sealed with hydrogen peroxide
It closes, then is closed with 5% normal serum, was then incubated at 4 DEG C with primary antibody LYVE-1 (Abcam, Cambridge, UK)
Night.Then the super streptavidin mark of 30min and horseradish peroxidase conjugation are incubated for the secondary antibody of biotin labeling
Remember reagent 30min.It is developed the color with 3,3'-diaminobenzidine (DAB) solution.
LYVE-1 and podoplanin is detected to the specific stain of intestinal cancer frozen tissue
When mice tumors grew to diameter is 0.8-1.0cm, mouse is put to death, by tumor tissues liquid nitrogen flash freezer 10-20
It is placed on after s spare in -80 DEG C of refrigerators；Frozen tissue section is prepared, fixes 10min with ice acetone；Frozen tissue section is soaked
Steep the 10min in PBS solution；Slice is taken out, different antibody is added dropwise on the position of adhering tissue, concentration is 1 μ g/mL, is allowed
It is completely covered organizationally, is incubated for 30min at room temperature；Slice is taken out, is immersed in PBS and is washed 3 times；Containing swollen
DAPI dye liquor is added dropwise in the position of tumor tissue, makes it that tissue be completely covered, is incubated for 5min at room temperature；By slice PBS washing by soaking 3
Time, anti-fluorescence quenching is added dropwise after to be dried, with coverslip mounting；Tissue is observed under Laser Scanning Confocal Microscope.
5, magnetic bead sorting
2 4-5 week old scid mouse are taken, every inoculates CT26 cell in right side oxter, long to tumor average volume
To about 1.5*1*1cm3, tumor-bearing mice is obtained, 75% ethyl alcohol 3-5min is soaked in after the execution that dislocated, after being sterilized
Mouse cuts off mouse skin from the mouse chest after disinfection with sterile scissors and aseptic nipper, carefully removes in super-clean bench
The culture dish of PBS is put into tumor tissues, after accomplishing that mouse not bleeding sufficiently shreds immersion on sterilized petri dishes with eye scissors
Tumor tissues are added dropwise 0.01M PBS in the process shredded and keep tissue wet, the tissue shredded is drawn to Pasteur's dropper
In 50mL centrifuge tube, adds the clostridiopetidase A I of tissue three times volume, be then placed on vortice and shake 10min, after obtaining concussion
Clostridiopetidase A I- tumor tissues mixture be put into 37 DEG C of water-bath 20min, repeat five times.After digestion, complete medium is used
Clostridiopetidase A I is terminated, 2min is stood, 300g is centrifuged 10min, then is washed twice with PBS, adds 10mL PBS resuspension, then slowly fall
The screen filtration for entering 200 mesh, filtered cell is counted, and is averagely assigned in two 15mL centrifuge tubes, is resuspended in PBS
It is centrifuged 10min under 300g, washes repeatedly 3 times.(1) MACS magnetic bead sorting method: by the 100 μ L buffer weight of the cell after washing
It is outstanding, 10 μ LLYVE-1-PE antibody are added and mix, is protected from light at 4 DEG C and is incubated for 15min, are then washed three times with 2mLbuffer, centrifugation
Afterwards, 80 μ L buffer are added and cell is resuspended, add the anti-PE magnetic bead of 20 μ L, washed three times after being incubated for 15min, with 300 μ after centrifugation
Lbuffer mixed pillar, finally quickly laid positive cell in pillar with 3mL buffer.Obtained cell is put six
It is cultivated in orifice plate.(2)Fe3O4The sorting of@KCTS-LEC-Doubleantibody magnetic nanoparticle: the cell after washing is used
200 μ L buffer are resuspended, and the Fe being prepared is added3O4@KCTS-LEC-Double antibody magnetic nanoparticle is mixed
It is even, it is protected from light at 4 DEG C and is incubated for 20min, then washed three times with 4mL buffer, after centrifugation, 160 μ L buffer are added and are resuspended carefully
Then born of the same parents are eluted cell suction with magnet, be placed on six orifice plate cultures.
6, the cell purity after the sorting of Immunofluorescence test distinct methods
1. preparing 12 orifice plates, PBS first is dripped in 12 orifice plate bottom centers, then clean coverslip is put into 12 orifice plates
In, with tweezers light cap slide, it is made to be tightly attached to orifice plate；The cell that different method for separating are sorted adjusts cell with PBS respectively
Concentration is 5 × 104Cell is uniformly laid in 12 orifice plates by/mL, the cell suspension of every hole addition 1mL, and 37 DEG C, 5%CO2Lower training
It supports for 24 hours；12 orifice plates are taken out, microscopically observation cell state is inhaled when cell state is good and abandons old culture medium, washed with PBS
3 times；4% paraformaldehyde, 200 μ L is added in every hole, the cells are fixed at room temperature 20min；It is washed 3 times with PBS, is separately added into LYVE-
1 and podoplanin antibody is protected from light in 4 DEG C is incubated for 2h, is washed 3 times with PBS；The DAPI dye liquor of 50 μ L is added in every hole, at room temperature
It is incubated for 10min, to wash cell 3 times by nuclei dyeing au bleu, then with PBS；Prepare glass slide, mark cell type,
Anti- fluorescence quenching is dripped in glass slide center, the cover for being paved with cell is then placed in laser scanning on glass slide
It is observed under Laser Scanning Confocal Microscope.
Streaming
The cell and Mouse LYVE-1PE-conjugated Antibody (article No. that distinct methods are sorted
FAB2125P, R&D) and 488 (article No. of Podoplanin Monoclonal Antibody AlexaFluor
53538180, ThermoFisher Scientific) antibody is resuspended in 200 μ L combination buffer (10mM HEPES/ NaOH
PH 7.4,140mM NaCl, 2.5mM CaCl2) incubation 1h is protected from light in 4 DEG C, it is washed 3 times with PBS, then uses flow cytometer
(BDAccury6；Becton Dickinson, San Jose, CA, USA) and use FlowJo software (Tree Star Inc.,
Ashland, OR, USA) it is analyzed.
7, endothelial cell phagocytosis experiment
2 × 10 from distinct methods sorting are collected respectively5Cell is added in EP pipe and contains final concentration of 20 μ g/
The 500 μ L of complete culture solution of mL DiI-ac-LDL (Molecular Probes, Invitrogen) is inoculated in after piping and druming uniformly
It is covered in 24 orifice plates of coverslip, 37 DEG C of incubation 4h.It removes and washes three times with 0.01M PBS after old culture solution, at room temperature 4% poly
Formaldehyde (Beyotime Biotech, China) fixes 30min, after 0.01M PBS washes three times, 100ng/mL DAPI (Beijing
Solarbio Science&Technology Co.) working solution dyeing 3min after PBS sufficiently wash.It is dripped on glass slide
After anti-fluorescent quenching mounting liquid (Beyotime Biotech, China), by coverslip one containing cell facing towards glass slide
It places (attention prevents bubble from generating), is fixed under rear fluorescence microscope (Nikon, Japan.) and seen with resin glue around coverslip
It examines.
8, Matrigel is tested at pipe
The logarithmic growth phase cell from distinct methods sorting is collected respectively, and digestion counts, and adjustment cell concentration is 1
×106Cells/mL is put into six orifice plates.Cell is resuspended with specific Endothelial cell culture base EBM-2 (Lonza), is put into six
In orifice plate, adjustment cell concentration is 5 × 105A/mL.37 DEG C, 5%CO2Incubator culture 5 days.To the every Kong Zhongjia (50 of 96 orifice plates
~60) Matrigel of μ L, at room temperature stable 15min, in 37 DEG C of incubators, 30min.In the first six orifice plate of digestion
Cultured cell (described above), into 96 orifice plates for be covered with matrigel, every hole is spread into 103A cell.37 DEG C, 5%CO2Culture
It is incubated for 6h in case, observes and takes pictures at microscope (Nikon, Japan).
9, statistical method
Using 16.0 statistical software of SPSS.Measurement data indicates that more comparison among groups use variance analysis with x ± s；It counts
Data compares using χ2It examines.P < 0.05 is that difference is statistically significant.
As a result
Fe3O4The characterization of@KCTS-LEC-Double antibody
The ultra microstructure that transmission electron microscope (TEM) is usually used in observing substance can be used because it is with high imaging resolution
It is detected in the characterization of nano material, the particle size and degree of scatter of nano material is assessed with this.Compound appearance such as Figure 1A
It is shown, rounded under the observation of transmission electron microscope, uniform in size, no particle aggregation phenomenon, no breakage.Partial size point
Cloth is consistent, about 68.06-78.02nm.DLS measures Fe3O4@KCTS-LEC-Double antibody compound is hydrated partial size
98.49 ± 8.31nm (Figure 1B).Size distribution ranges are relatively narrow in water for nano particle as seen from the figure, good dispersion.Its surface
Zeta potential be -32.77 ± 1.51mV, surface elecrtonegativity (Fig. 1 C), absolute value be greater than 30, show that the stability of material is good.
Fluorescence spectrometer is as the result is shown (Fig. 1 D): antibody LYVE-1 (FITC) has obvious emission peak under 488 exciting lights,
Podoplanin (PE) also obviously has an emission peak under 545 excitations, and Fe3O4@KCTS-LEC-Doubleantibody compound
There is emission peak respectively under 488 and 545 exciting lights, shows that the material is coupled successfully.
Immunohistochemistry and immunofluorescence
Immunohistochemistry shows the high expression (Fig. 2 is left) of LYVE-1 in tumor tissues, frozen section show LYVE-1 and
Podoplanin antibody can express simultaneously the right side Fig. 2 in frost tumor tissues.It illustrates well in mouse Colon cancerous tissue
In have a small amount of lymphatic endothelial cells.
The separation and enrichment of LEC cell
The cell that two kinds of different method for separating obtain is carried out streaming interpretation of result such as Fig. 3 A by us, through LYVE-1
The coexpression rate of cell LYVE-1 and Podoplanin that MicroBeads magnetic bead sorting obtains are 71.2%, and through Fe3O4@
Obtained two kinds of marker coexpression rates of cell are after the sorting of KCTS-LEC-Doubleantibody magnetic nanoparticle
88.9%.The result shows that through Fe3O4The cell purity obtained after the sorting of@KCTS-LEC-Doubleantibody magnetic nanoparticle
It is higher, to more reliable practical about tumor lympha endothelial cell correlative study.
Matrigel glue is tested at pipe
Matrigel main component has IV collagen type, laminin, heparin sulfate glycoprotein, nestin, also wraps
Include matrix metalloproteinase and growth factor etc..At normal temperature, Matrigel is polymerizable forms the three-dimensional with biological function
Matrix, the composition of cell basilar memebrane, structure and function in analog body, are conducive to cell culture in vitro and differentiation can also be right
The form of cell, the structure and function of endothelial cell, invasion, migration and gene expression research.Through LYVE-1MicroBeads
Magnetic bead sorting obtains and is inoculated into the cell on Matrigel forming capillary sample pipe structure in 12 hours, through Fe3O4@KCTS-
The cell obtained after the sorting of LEC-Doubleantibody magnetic nanoparticle forms more multiple capillary sample pipe structure.It confirms pure
It is stronger at pipe ability to spend higher lymphatic vessel.
Endothelial cell phagocytosis experiment
Dil-ac-LDL is a kind of acetylated low density lipoprotein, is marked with Dil red fluorescence object, it and endothelial cell table
Face receptor is swallowed after combining.After endothelial cell absorbs ac-LDL, it can assemble on lysosome membrane.It can be used as endothelial cell characteristics
The phenotype of property.Dil-ac-LDL intake measurement display, the cell obtained through LYVE-1MicroBeads magnetic bead sorting are displayed in red
Fluorescence, and through Fe3O4The cell obtained after the sorting of@KCTS-LEC-Doubleantibody magnetic nanoparticle shows stronger
Fluorescence (Fig. 3 D).
It discusses
It for a long time, is considered as the important channel of tumour cell diffusion by the transhipment of lymphatic system, however as leaching
Abundant research is not yet received compared with the vascular endothelial cell (BEC) in its blood vessel in the LEC of hand shaft main component.LEC label
Identification promotes the research to LEC, and helps to distinguish them with BEC.LYVE-1 (Lymphatic vessel
Hyaluronan receptor-1) be a kind of I type that 322 amino acid residues is made of of the expression on cell membrane completely across
Membrane glycoprotein, and be considered as lymphatic endothelial cells mark most special at present.There is experiment to confirm that LYVE-1 expression exists
On the lymphatic endothelial cells of a variety of different tissue sources.Podoplanin (glomerulus progenitor cell surface albumen) is I type cross-film
Saliva mucin sample glycoprotein, molecular weight 38Kda, Podoplanin are not the exclusive marker of lymphatic endothelial cells
But the expression on skin in the blood vessels is not yet found, so often thin to verify lymphatic endothelial in conjunction with other lymphatic vessel markers
Born of the same parents.This research has selected the combination of the two antibody then to sort LEC cell, and by immunofluorescence common location, the double dyes of streaming are altogether
The methods of expression sorts the purity of cell to verify.The cell of the two antibody of energy double expression more meets lymphatic endothelial cells
Biological characteristics, stronger at pipe ability, phagocytic activity is more preferable.
Recently, the purification process based on immune magnetic is used to separate different cells in weight mixed culture, with LYVE-1 or
The coated magnetic bead of podoplanin antibody is respectively used to separate relevant subgroups and granular cell.These methods are only trained from several mixing
It supports and has purified specified subgroup in object, therefore this does not only result in the omission for the part LEC for initially including, and leads to foreign cell
Such as the pollution of vascular endothelial cell, and spend the time long.Before reporting several new specific lymph markers, LEC master
It will be by being separated in the never same tissue of collagen enzymatic treatment.But during originally culture, the processing of clostridiopetidase A may also lead to other
The pollution of cell, including vascular endothelial cell and interstitial cell.Herein, we combine by specificity target molecule such as polypeptide,
The modifications such as antibody, aptamers apply the enlightenment on diagnosing tumor and treatment, we optimize mesh to magnetic Nano material surface
The antibody of preceding maturation is coated with magnetic bead sorting method, devises a kind of KCTS coated magnetic Fe3O4Nano particle, being formed has nucleocapsid
The Fe of type3O4@KCTS nano particle, partial size is small, good dispersion, can have hydrophilic target in its surface modification easily
To molecular antibody, magnetic Fe is successfully constructed3O4@KCTS-LEC-Doubleantibody nano target probe, particle size is about
For 100nm, transmission electron microscope shows good dispersion, and not easy to reunite, after antibody modification, increased hydrophilicity is uniformly dispersed, raw
Object compatibility increases.By CCK-8 toxicity test, influence of the nano-probe to cell viability is verified, as the result is shown Fe3O4@
KCTS-LEC-Doubleantibody nano-probe does not have toxicity to LEC cell.
This research successfully constructs magnetic Nano probe, can pass through bio-safety in conjunction with the target molecules of specificity
The verifying of property, is applied to sorting lymphatic endothelial cells, and compare with the coated magnetic bead of common monoclonal antibody, double
The method of antibody coating self-control magnetic bead substantially increases the purity of LEC, reduces the sorting time, improves cell activity, reduce
Cost, this for later tumour through lymphatic vessel shift and lymphatic endothelial cells mechanism research provide it is valuable experiment according to
According to, and magnetic Nano material can be applied to NMR imaging, and also the targeting diagnosis for later clinical cancer provides valuable
The Research Thinking of value.
Claims (10)
1. a kind of magnetic probe, comprising: hud typed Fe3O4@KCTS magnetic nanoparticle and modification are described hud typed
Fe3O4The lymphatic endothelial cells double antibody on@KCTS magnetic nanoparticle surface；
The lymphatic endothelial cells double antibody is LYVE-1 antibody or its antibody fragment and Podoplanin antibody or its antibody
Segment；
Wherein, KCTS indicates α-ketoglutaric acid contracting chitosan.
2. magnetic probe according to claim 1, it is characterised in that: the partial size of the magnetic probe is 80-150nm；Point
Dissipating sex index is 0.2-0.5.
3. the method for preparing magnetic probe of any of claims 1 or 2, comprising:
1) chitosan is subjected to carboxylated modification with α-ketoglutaric acid, obtains the α-ketoglutaric acid contracting shell that surface is rich in-COOH base
Glycan KCTS；
2) under the activation of carbodiimide, Fe3O4Magnetic nano-particle passes through covalently bonded by the-COOH of surface-OH and KCTS
It closes, obtains Fe3O4@KCTS magnetic nanoparticle；
3) two kinds of LEC antibody are covalently bonded to the Fe that step 2) obtains3O4@KCTS magnetic nanoparticle surface, obtains Fe3O4@
KCTS-Double antibody magnetic nano-particle, i.e. magnetic probe；
In step 3), described two LEC antibody are LYVE-1 antibody or its antibody fragment and Podoplanin antibody or its antibody
Segment.
4. according to the method described in claim 3, it is characterized by: in step 2), KCTS and Fe3O4The matter of magnetic nano-particle
Amount is than being 1-5:1.
5. the method according to claim 3 or 4, it is characterised in that: the operation of step 3) are as follows: by Fe3O4@KCTS magnetism is received
Rice grain is dissolved in HEPES buffer solution, ultrasonic disperse, and EDC/NHS crosslinking agent is added, and is incubated for, after having reacted, magnetic separation goes out
The Fe of crosslinking activation3O4@KCTS；By two kinds of LEC antibody, i.e. LYVE-1 antibody or its antibody fragment and Podoplanin antibody
Or its antibody fragment, it is added separately to the Fe of above-mentioned crosslinking activation3O4It in@KCTS, mixes, is incubated for；Add bovine serum albumin
Closing；Magnetic separation obtains Fe3O4@KCTS-Double antibody magnetic nano-particle.
6. according to the method described in claim 5, it is characterized by: LYVE-1 antibody or its antibody fragment, Podoplanin are anti-
Body or its antibody fragment and Fe3O4The proportion of@KCTS magnetic nanoparticle successively may be used are as follows: 1-10 μ g:1-10 μ g:1mg；
It is 1-3h that the time being incubated for after antibody, which is added,.
7. magnetic probe of any of claims 1 or 2 is that is, Fe3O4@KCTS-Double antibody magnetic nano-particle is drenching
Application in the sorting of hand shaft endothelial cell and identification.
8. a kind of sorting/identification lymphatic endothelial cells method, comprising:
By magnetic probe of any of claims 1 or 2, i.e. Fe3O4@KCTS-Double antibody magnetic nano-particle with to
It sorts mixing with cells to be incubated for, centrifugation, buffer is added into the solid precipitating being collected into, cell is resuspended, with magnet by cell suction
It elutes.
9. according to the method described in claim 8, it is characterized by: the temperature of the incubation be 4-37 DEG C, time 15-
60min。
10. sorting/identification method of the lymphatic endothelial cells of claim 8 or 9 is in lymphatic endothelial cells sorting/identification
In application.
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