CN107349636A - Capillary and its preparation and application of the biomaterial as interaction phase - Google Patents
Capillary and its preparation and application of the biomaterial as interaction phase Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/20—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the conditioning of the sorbent material
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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- B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
- B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
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Abstract
The invention provides it is a kind of sieved with post and using biomaterial as the interaction capillary of phase, its preparation method and purposes by capillary electrophoresis method in research large biological molecule and active ligand or compound phase interaction and drug screening.Difficulty is isolated and purified from cell or tissues in vitro greatly and can cause the change of native conformation for some bioactive macromolecules, the present invention develop by biomaterial (such as large biological molecule overexpression cell, cell, mitochondria, in vitro tissue, tumor tissues, carry biological target solid matter etc.) inject in the capillary with post sieve, the biomaterial is intercepted in post sieve rear end as interaction phase, establish on-fixed metaplasia thing material capillary electrophoresis tube (Non immobilized Biomaterial Capillary Electrophoresis, NIBCE), close under the conditions of physiological environment, for studying the capillary electrophoresis method of large biological molecule and active ligand or compound phase interaction and drug screening etc..
Description
Technical field
The present invention relates to capillary and its preparation method and application, more particularly to for intercepting as interaction phase
Biomaterial post sieve capillary chromatographic column, its preparation method and capillary chromatographic column pass through HPCE
(High Performance Capillary Electrophoresis, HPCE) is in biomedicine field for example in research biology
The application of macromolecular and active ligand or compound phase interaction and drug screening etc..
Background technology
Most of biomolecule are all to be interacted by the way that specificity occurs with its target molecule to play its physiological function,
Such as enzyme and substrate, antigen and antibody, acceptor and hormone, agglutinin and GL-PP etc..These specificity being of interaction
The phenomenon with being widely present in biochemistry system is learned, even more produces the basis of biological phenomena.Biomolecule and its function are matched somebody with somebody
As one of emphasis of biochemical research, this helps to go deep into various between understanding large biological molecule the sign of body phase interaction
The architecture basics and mechanism of action of function, contribute to the discovery of new drug.Therefore, between developmental research biomolecule or large biological molecule
Become particularly important with the method for other ligand-ligand interactions.
It is big that the method for the biological intermolecular or large biological molecule of research and other ligand-ligand interactions can be largely classified into two
Class：One kind is direct measuring method, including UV absorption processes, Raman spectroscopy, fluorescence intensity method, potentiometry, thermal analysis system, nuclear-magnetism are common
Method of shaking (NMR), fourier transform infrared spectroscopy (FTIR), mass spectrography (MS), Surface Plasmon Resonance (SPR) etc.；It is another
Class is segregation analysis, including Filter assay method, ultrafiltration, supercentrifugation, chromatography (thin-layered chromatography and liquid chromatogram
Method), electrophoresis (disk electrophoresis method and HPCE).The advantages of the latter is：Two kinds of molecules of interaction can be simultaneously provided and answered
The information of compound, avoid the interference of coexisting substances.Filter assay method included by segregation analysis, ultrafiltration, ultracentrifugation
Method, chromatography and disk electrophoresis method be frequently utilized for study ligand-receptor between interaction, but they exist binding molecule from
Seepage on film, stereomutation, Donnan effects, non-specific adsorption, the shortcomings of analysis time is long, sample consumption is big；In recent years,
Technology based on HPCE turns into the biological intermolecular or large biological molecule of research and the main side of other ligand-ligand interactions
Method.
HPCE is used to study biological intermolecular or large biological molecule and other ligand-ligand interactions, has advantages below：
Separative efficiency is high, up to 105-106Plate/rice；Analysis time is short, can complete within tens seconds to more than ten minutes to analyze；Sample consumption
Few, volume needed for sample introduction can be as small as 1 μ L, and consumption volume is between 1-50nL；Can be in physiological condition or close to physiological condition
Carried out in buffer solution, can obtain the information of the interphase interaction of more real biomolecule；Different kinds of molecules can be studied simultaneously
To a kind of bonding behavior of molecule；Use flexibly, different patterns can be selected to be applicable different separation objects；It is economical；It is clean
Only；Automaticity is higher.Wherein, the form that HPCE is used to study interaction has pre-equilibration zone electrophoresis, kinetic balance
Zone electrophoresis and immobilization affinity electrophoresis.There is in recent years and successively immobilized cell capillary electrophoresis (ICCE), cell membrane
Chromatography (cell membrane chromatography, CMC) so that the acceptor that can not be separated can directly by HPCE or
CMC carries out ligand screening.Cell is fixed in chromatographic column and carries out receptors ligand repercussion study and is applied to drug screening
It is the new technology developed in recent years, the advantage that the technology protrudes is to inseparable being fixed of cell-membrane receptor, from
And overcome the problems such as accuracy rate existing for traditional biological method is low, toxicity is big, cost is high.Immobilized cell technique utilizes physics
Or cell is fixed on suitable carrier by chemical method, make its play bioactivity, it is easier than enzyme immobilization method, quick,
It is economical.But this method still has complex operation, it is difficult to prepare, and cycle length, service life is short, and high expressed receptor cell can only be carried out
The shortcomings of screening.
Therefore, on the basis of ICCE, method is innovated, foundation can to large biological molecule overexpress (such as
The various biomaterial tables such as cell GLUT1), cell, mitochondria, in vitro tissue, tumor tissues, carrier band biological target solid matter
The method that face acceptor directly screens becomes particularly important.
The content of the invention
It is (i.e. non-solid as capillary, the capillary electrophoresis system of interaction phase the invention provides a kind of biomaterial
Determine metaplasia thing material capillary electrophoresis tube, NIBCE) and its preparation and use.
The present invention provides a kind of new capillary, prepared with the controllable post sieve in aperture first, and injects by solid
Fixed biomaterial is as interaction phase, and the acceptor of the interaction phase surface keeps native conformation and bioactivity, and
And it have studied and this new capillary is such as studied into large biological molecule and active ligand by HPCE in biological technical field
Or the purposes of compound phase interaction and drug screening etc..
One aspect of the present invention provides a kind of capillary, its phase of sieving and interact with post, wherein the hole of post sieve
Footpath is controllable, available for the biomaterial intercepted as interaction phase.
The post sieve may be provided at the middle part or end of the capillary, or post sieve is connected to the capillary
The end of pipe.
The biomaterial includes cell, mitochondria, tissues in vitro, tumor tissues, carrier band biological target solid matter
Deng.Wherein described cell is the cell or tumour cell of large biological molecule overexpression, and the macromolecular acceptor of cell surface is high to express,
And keep native conformation and bioactivity.
The controllable post sieve in aperture is provided with the capillary or between capillary, biomaterial is injected into capillary after fixation
It is interior to be used as interaction phase, the acceptor of the interaction phase surface is kept native conformation and bioactivity.Wherein described post sieve
Material be inorganic material, organic material, composite, biomaterial or other any applicable materials.The opposing party of the present invention
Face provides a kind of method for preparing the capillary, and it includes：
Pre-treatment is carried out to capillary；
Post sieve material is pre-processed and forms post sieve, make post sieve be arranged on the capillary middle part or
End, or the end for making the post sieve be connected to the capillary, so as to form the capillary with post sieve；If the post sieve
It is arranged on the end of the capillary or is connected to the end of capillary, then needs the end of capillary and there is detection window
The end of capillary be attached by connector；
To being cultivated or being handled and is fixed as the biomaterial of interaction phase；And by described in after fixation
In the biomaterial injection capillary with post sieve so that the biomaterial is intercepted sieves rear end as phase interaction in post
With phase.
In addition, the invention further relates to a kind of HPCE systems, it includes：Capillary of the present invention with the controllable post sieve in aperture
Pipe；For by the device of biomaterial and Sample introduction capillary；Electrophoresis behaviour is carried out to the sample for importing the capillary
The device of work；And to device that the sample is detected.
Meanwhile present invention also offers a kind of HPCE methods, it includes providing the capillary with the controllable post sieve in aperture, and
Capillary is injected after the biomaterials such as cell, mitochondria, in vitro tissue, tumor tissues are fixed as interaction phase；There is provided
Electrophoretic apparatus；By capillary described in biomaterial or Sample introduction；HPCE analyses are carried out to the sample；And to the sample
Tested and analyzed.
The HPCE methods are applied to, such as studying the Capillary Electrophoresis of intermolecular interaction, including array hair
Cons electrophoresis, chip capillary cataphoresis, array chip Capillary Electrophoresis, capillary liquid chromatography etc..
Further, present invention also offers above-mentioned capillary or capillary chromatographic column biological technical field purposes.
The biological technical field includes, such as drug screening, large biological molecule and active ligand interaction or mutual with compound
Effect, drug ingedient analysis, active constituents of medicine purifying and identification etc..
The post sieve that the present invention is established intercepts biomaterial (cell, mitochondria, in vitro tissue, tumor tissues after fixing
Deng) as interaction phase, the method for studying intermolecular interaction and drug screening, can be used in compound with by
Body phase interaction carries out qualitative, quantitative research.Compared with ICCE and other capillary inner cell process for fixation, institute of the present invention
The advantages of offer method, is：(1) when cell etc. is as interaction phase, the cell for injecting capillary has dynamic and stable state
The interaction of medicine and tumour cell that two states, wherein current intelligence can be used in simulation physiological environment；(2) phase interaction
Capillary can be got by counter with phase, updated at any time, the false negative result for avoiding avtive spot from being saturated and bringing, capillary
Post may be reused, degree of recycling height；(3) biomaterial is intercepted and can not reach detector, so as to eliminate it to inspection
The interference of device is surveyed, is particularly conducive to quickly find the material for having combination from mixture using HPCE-MS, and pass through binding constant
The most strong material of calculating selection binding activity；(4) amount of samples is few, high sensitivity, favorable reproducibility, and chromatographic peak profile is preferable, can
Receptors ligand is interacted using NLC technologies and carries out quantitative analysis, provides kinetic parameter K, k ', ka, kd；(5) preparation method
Very simple, quick, cost is low, and experimental period is short；(6) this method has widened application, and different biomaterials is introduced
Capillary is as interaction phase, for studying intermolecular interaction, with more actual application value.
The present invention sieves biomaterial (cell, mitochondria, in vitro tissue, the tumor group intercepted in injection capillary by post
Knit) and the immobilization of the macromolecule receptor protein to being difficult to isolate and purify is realized, it furthermore achieved that to macromolecule acceptor
The affinity chromatography ACE researchs of protein.Secondly, HPCE possesseds are quick, efficient, economic, sensitive, sample because having for the method
The advantages that dosage is few, it can be mixed thing or single compound and biomaterial repercussion study and be different from other drugs
Screening technique.Some currently used drug screening technologies are only capable of providing IC50Value, and the method for the present invention can provide binding constant
Deng kinetic parameter.
Capillary and correlation technique of the present invention are also applied for other field and have obvious technical advantage, such as：
Capillary array electrophoresis high-flux medicaments sifting method, screen the pro-drug in combinatorial chemical library or traditional Chinese medicine.
Brief description of the drawings
Fig. 1 shows that the preparation sieved according to the capillary column of one embodiment of the present invention (sieves material using silica gel as post
Exemplified by) and the related figure established of screening technique.Wherein：A be capillary column sieve electron-microscope scanning figure (SEM, JSM-5600LV,
JEOL,Japan)；B is that the GLUT1-eGFP gathered by Laser Scanning Confocal Microscope transfects the image after the cell 36h of HEK 293
(GLUT1-eGFP is positioned at cell membrane, and shown in green fluorescence, the nuclear staining of GLUT1-eGFP HEK293 cells is blue glimmering
Light)；C is the image of the immobilized cell chromatographic column gathered by Laser Scanning Confocal Microscope；In the capillary of D light microscopes collection
Cell state image (left side is " dynamic " cell, and the right side is " stable state " cell)；E is under different GLUT1-HEK293 cell densities
EGCG HPCE figures；F is DMSO, EGCG HPCE figures under dynamic and static GLUT1-HEK293 cell states；According to G
The present invention establishes on-fixed metaplasia thing material capillary electrophoresis tube (Non-immobilized Biomaterial Capillary
Electrophoresis, NIBCE), (post sieve capillary, common HEK293 are thin on different capillary chromatographic columns by DMSO and EGCG
Born of the same parents as interact phase, overexpress GLUT1 HEK293 cells as interact phase) electrophoretogram.
Fig. 2 is the dynamics determined according to the embodiment EGCG of the present invention by two methods of NICCE and ICCE
Parameter comparison sheet.
Fig. 3 is screened for the radix scutellariae total flavone extract (A) screened according to the embodiment of the present invention and part
The HPCE figures of compound (B), and the glucose competitive assay electrophoretogram (C) of the amount of activated compound filtered out.
Fig. 4 lists the structure for 22 compounds screened according to the embodiment of the present invention.
Fig. 5 is the selection result and NICCE the and ICCE result tables of comparisons of 22 compounds described in Fig. 4.
Part of compounds when Fig. 6 is according to embodiment A549 tumour cell of the invention as interaction phase
HPCE schemes.
Fig. 7 is part when being used as interaction phase according to the in vitro lung tissue of embodiment and tumor tissues of the present invention
The HPCE figures of compound.
Fig. 8 is EGCG, FSB-1, FSB-2, FSB-3, SL-3, SL-4 MTT cellular level antitumor activity experimental results.
Fig. 9 is the horizontal antitumor activity experimental result of animal.Wherein：A, when B is that animal was put to death in the 27th day after being administered, physiology
The animal tumor growth of salt solution group (NS groups), EGCG groups (80mg/kg), FSB-1 groups (80mg/kg) and FSB-2 groups (80mg/kg)
Situation；C is each group the weight of animals situation of change after administration；D is each group animal tumor Volume Changes situation after administration；E is administration
When animal is put to death within the 27th day afterwards, the weight of each group tumour.* represent has significant difference (* P compared with NS groups<0.05,**P<
0.01,***P<0.001)。
Embodiment
Further, exemplary description is made to the present invention below in association with some specific embodiments, is included in
Prepare post sieve in capillary or between capillary, the culture and fixation of cell, the extraction of mitochondria, in vitro tissue and tumor tissues
It is fixed, the biomaterials such as the cell, mitochondria, in vitro tissue, tumor tissues are injected in capillary as interaction phase
Method, and this new capillary by HPCE biological technical field such as research large biological molecule match somebody with somebody with activity
The purposes of body or compound phase interaction and drug screening etc..
In a kind of embodiment included by the present invention, by the silica gel powder of a kind of material powder, such as certain particle diameter,
After fully being handled with solution, capillary midsection or end are packed into, after heating certain time, prepares pH tolerance ranges
Extensively, the post sieve capillary that reappearance is good, aperture is controllable.
In one embodiment, by biomaterial, such as the biological material such as cell, mitochondria, in vitro tissue, tumor tissues
In capillary chromatographic column of the material injection with post sieve, so as to obtain with capillary chromatography of the biomaterial as interaction phase
Post.
In one embodiment, by a kind of cell, such as ordinary cells, the cell of large biological molecule overexpression, tumour
Cell, injected after fixation in capillary, so as to obtain with capillary of the cell as interaction phase.
In one embodiment, will pass through appropriate culture (such as culture mediums of RPMI 1640 containing 10-20%FBS,
At 37 DEG C, 5%CO2Cultivate in incubator, its incubation time can be such as 10-48h) cell, the cell after electricity turn is placed in PBS
Middle suspension cell to density is, for example, 1.0 × 104-1.0×107Individual/mL, such as 4% poly first is then used to living cells layer
Aldehyde is handled, and its processing time can be such as 5-25min.
In one embodiment, by vitro tissue, tumor tissues, handled using 4% paraformaldehyde, during processing
Between can be such as 12h-48h, be stored in after processing standby in 75% ethanol.
While above-mentioned novel capillary preparation method is provided, embodiments of the present invention are also included the capillary
Chromatographic column is used for the biotechnology such as operating method including drug screening.In one embodiment of the invention, according to spy
Fixed operating condition, it is that negative control has carried out method validation by positive control, dimethyl sulfoxide (DMSO) (DMSO) of EGCG.As a result table
Bright, EGCG and DMSO is overexpressing the color in capillary chromatographic column of the GLUT1 HEK293 cells as interaction phase respectively
Spectrum behavior is entirely different, can qualitatively judge accordingly between compound and acceptor with the presence or absence of interaction.
A kind of embodiment provided by the present invention also includes the capillary being used for HPCE system and method.It is described
HPCE methods include：Capillary of the present invention is provided, in-between position or end set have post sieve, and intercept as mutual
Act on the biomaterial of phase；Electrophoretic apparatus is provided；By capillary described in biomaterial or Sample introduction；Electricity is carried out to the sample
Swimming analysis；And the sample is tested and analyzed.
In other embodiments of the present invention, for phase that is true, reliably and quickly reflecting acceptor and part
Interaction, the present invention establishes to intercept after the HEK293 cells fixation for overexpressing GLUT1 acceptors sieves rear end as phase interaction in post
With the method for mutually studying intermolecular interaction.And for flavones monomeric compound, strepto- in radix scutellariae total flavone extract, radix scutellariae
Pseudomonas metabolite, bacillus metabolite have carried out combining screening active ingredients.On this basis, in order to further verify institute
The interaction of the active material filtered out and actual tumour cell and tumor tissues, the present invention establish A549 tumours first
Method after cell, in vitro lung tissue, the fixation of A549 cell transplantations tumor tissues as interaction phase, is combined for studying
Reactive compound and their interaction.
In other embodiments of the present invention, in order to judge between positive compound and glucose with the presence or absence of competition
Property combine, the present invention is that target spot carries out positive compound and glucose competitive binding experiment using GLUT1, such as in PB buffer solutions
The middle glucose for adding different quality respectively is configured to the dextrose buffer liquid containing 0-10mM, and observation positive compound is slow at these
HPCE Behavioral changes in fliud flushing, the binding site of glucose whether is occupied during so as to judge that they are combined with GLUT1.
In addition, using non-linear chromatography theoretical (Non-linear Chromatography, NLC) to the activity that filters out
Compound has carried out the quantitative calculating of binding constant, association rate constant, dissociation rate constant and capacity factor measure, while to this
A little compounds have carried out MTT experiment and have carried out cellular level antitumor activity, and compound further strong to activity (including
Competitive binding is not present with glucose and the compound of competitive binding be present) carry out the horizontal antitumor activity of animal.
As a result show, the reactive compound filtered out by this method can suppress the growth of tumour cell to some extent, be swollen to be anti-
Tumor medicine screens and oncotherapy lays the foundation and provides scientific basis and new approaches, also indicates that the most newly-established side of the present invention
Method is used for the bioactivity of screening at molecular level compound, and the ICCE methods of invention foundation are used for screening at molecular level chemical combination with early stage
Thing result is basically identical and effective.
Embodiment
With reference to specific embodiment, the present invention is furture elucidated.It should be understood that these descriptions are not intended to limit this Shen
The scope that please be claimed.
The HPCE new methods of embodiment 1, biomaterial as interaction phase, i.e. on-fixed metaplasia thing material capillary electricity
The foundation of swimming (NIBCE) (exemplified by using GLUT1 overexpression HEK293 cells as interaction phase)
The preparation of capillary column sieve and performance verification：Long 30.2cm, 200 μm of I.D. vitreous silica capillary are used successively
Methanol, ultra-pure water, 0.1M HCl, ultra-pure water, 0.1M NaOH, ultrapure water, after drying by a certain amount of silica gel, SE-30 or
ODS powder prepares bonding post sieve by heating in capillary, obtains the capillary with post sieve, room temperature preservation is standby.Silica gel
The electron-microscope scanning figure of the capillary column sieve prepared as raw material is shown in Fig. 1 (A).
The performance verification of capillary column sieve：In the present embodiment, HPCE used is Beckman P/ACETM MDQ systems
(Beckman-Coulter, Fullerton, CA, USA), equipped with a two level array detector and 32KaratSoftware
Work station (version 5.0, Beckman), outer layer are the fused quartz capillary chromatography with post sieve of polyimide coating
Post.PH tolerance ranges that this experiment is sieved to post, post sieve reappearance are verified.This experimental formula difference pH NaOH or HCl
Solution, sample introduction feminine gender compound DMSO, twice among sample introduction respectively with alkali or acid recoil 5-10min, sees under the same conditions
The change of DMSO appearance situations is examined, to judge the pH tolerance ranges of post sieve.Every group is repeated twice, and as a result reappearance is preferable.As a result
Prove, it is 1-14 to test prepared capillary column and sieve be resistant to pH scopes.In addition, three capillarys are prepared under the same terms
Tubing string sieves, and the sample introduction DMSO in continuous five days, carries out reappearance investigation to it respectively.Wherein, retention time in a few days, in the daytime and
The RSD of betweenrun precision is respectively 4.90%, 6.56%, 9.04%, peak area in a few days, in the daytime and batch between RSD be respectively
2.50%th, 4.13%, 6.77%, show the reappearance of prepared post sieve capillary with having good stability.
GLUT1 overexpresses the culture of HEK293 cells：The cell culture of HEK 293 containing 10% hyclone (FBS,
Canada GIBCO) the culture mediums of RPMI 1640 in, wherein containing penicillin (100U/ml) and streptomysin (100 μ g/ml).Will
The cells (4 × 10 of 400 μ l HEK 2936) by electric pulse generator (Electro Square PoratorECM830, BTX,
San Diego, CA), under the conditions of voltage 123V, burst length 20ms, instantaneously turn GLUT1-eGFP/pcDNA-DEST47 plasmids
10-20μg.The distribution of green fluorescence on fluorescence microscope HEK293 cell membranes, the visible figure of concrete condition are utilized after 36-48h
1 (B), it was demonstrated that most cells surface all expresses GLUT1.
GLUT1-HE293K cells fix after as interact phase capillary chromatographic column preparation：(t- after electricity is turned
After GLUT1-HEK293 cells 36-48h) digest centrifugation from culture dish, cell count, fixed with 4% paraformaldehyde lucifuge
After 15-25min, cell density is adjusted to 1.0 × 10 with PBS according to count results4-1.0×107Individual/mL.It is stored in 4 DEG C of ice
Case is stand-by.During experiment, the cell after above-mentioned fixation is injected into capillary, due to the presence of post sieve, these GLUT1 overexpressing cells
There are two kinds of existence forms in injection capillary：" dynamic " and " stable state ", i.e. cell have just been injected thin in a period of time in capillary
Born of the same parents do not reach the motion state of sieve plate rear end also, and cell total movement is shown in Fig. 1 (D) to the stable state of sieve plate rear end.This side
After the fixation of 4% paraformaldehyde, 4 DEG C preserve that still to remain within 30 days drug screening active in PBS for cell in method, and cell can be with
Reinjected during use in capillary, it is counter after use to get capillary, it is simple to operate, update at any time, avoid avtive spot quilt
Saturation and the false negative result brought, repeated recycling utilize degree are high.
The influence of varying number, the cell of different conditions and living cells to combination：The present invention has investigated different numbers
Measure cell, the influence of different conditions cell and living cells to combination.If Fig. 1 (E) is it can be found that EGCG is thin in different densities
Peak shape is varied from the capillary chromatographic column of born of the same parents.When cell density is 104With 105During individual/mL, EGCG peak height is only shown
Certain reduction, broadening unobvious；And as cell density increases to 106Individual/mL, EGCG peak shape show significant hangover exhibition
It is wide；When increasing to 107Individual/mL, peak shape show as being attached to a wide calm peak shape on baseline.As a result show, for there is knot
The compound of cooperation, it is as a result more genuine and believable when cell number reaches certain density.Using GLUT1 as target spot, DMSO is contrasted
With electrophoresis behaviors of the EGCG under different cell states, judge whether cell state has an impact to binding constant.Fig. 1 (F) is visible,
NICCE method is all suitable for for cell dynamic and stable state, and strong positive compound is more suitable for being tested in cell dynamic.Need
Will it is emphasized that under cell current intelligence more can the environment of aids drug in blood, the binding constant of gained is closer
In physiological condition.In subsequent experimental, no special instructions, all using being tested under cell dynamic.
In addition, the present invention has also investigated whether living cells under this methodology can not be fixed, directly carry out intermolecular mutual
The research of effect.As a result show, when living cells is as interaction phase, unstability of base line, peak shape are irregular.Therefore, this method
It is not suitable for the intermolecular interaction of living cells (cell fixed without 4% paraformaldehyde) research.
The HEK293 cells for overexpressing GLUT1 are used for method for screening compound as the capillary chromatographic column of interaction phase
Checking：GLUT1 transhipment substrate has glucose, galactolipin and mannose, but the sensitivity of three is low, therefore, in this experiment
Middle using EGCG as positive compound, DMSO is negative compound, have studied it respectively and sieves capillary, normal HEK 293 in post
Cell overexpresses the cells of HEK 293 as the color in the capillary of interaction phase as capillary, the GLUT1 of interaction phase
Spectrum behavior, shown in experimental result such as Fig. 1 (G).Compare electrophoresis graph discoveries of the DMSO in different chromatographic columns, its peak height peak shape does not have
Significant difference.And responses of 100 μM of the EGCG on post sieve capillary nearly reaches 25mAU, but with concentration sample introduction to injection
When in the capillaries of ordinary cells, the response at peak show it is significant reduce hangover broadening, this show EGCG with it is common thin
Non-specific interaction between born of the same parents be present；In capillary of the GLUT1 cells as interaction phase is overexpressed, 100 μM
EGCG shows as being attached to a wide calm peak shape on baseline, stronger specificity interaction be present.Equally, we adopt
Comparative study has been carried out with the ripe ICCE methods having built up early stage, the capillary chromatographic column that ICCE methods use is shown in Fig. 1 (C),
Electrophoresis behaviors of the DMSO and EGCG respectively on the different capillary columns in two methods is basically identical, further demonstrates this hair
The feasibility of bright methods described.
The change of chromatographic peak profile in affinity chromatography is the specific and non-characteristic phase interaction between compound and stationary phase
Result.It is higher than association rate generally, due to the dissociation rate of compound and stationary phase so that chromatographic peak shows as broadening,
The non-gaussian peak shape of hangover.In the case where chromatogram capacity is certain, compound peak shape deviates the degree and compound of Gaussian peak
Concentration is related.And NLC theories just explain the relation of concentration and peak shape change and the computational methods of various kinetic parameters.
The present invention carries out electrophoresis to EGCG as the capillary chromatographic column of interaction phase by the use of the HEK293 cells of GLUT1 overexpressions and divided
Analysis.The asymmetric peak shape of gained can be explained with non-linear chromatography model, and be fitted peak shape, calculations incorporated constant with software
(K), association rate constant (ka), dissociation rate constant (kd) and capacity factor measure (k ').By this method acquired results and ICCE methods
Acquired results are respectively calculated and compared, and are specifically shown in Fig. 2, it can be seen that, the EGCG and GLUT1 of this method measure combination are moved
Mechanics parameter result and ICCE methods are basically identical.
Embodiment 2, the HEK293 cells of GLUT1 overexpressions sieve as the capillary chromatographic column of interaction phase in compound
The application chosen
The screening medicine new method that the present invention is established is primarily used for screening radix scutellariae total flavone extract, as a result sees Fig. 3
(A).The chromatographic behavior result of extractive of general flavone is shown, when the HEK293 cells of GLUT1 overexpressions are as interaction phase,
Key component retention time delay in extractive of general flavone, peak height is reduced, and the peak shape of some components is seriously trailed, to enter one
Step finds which kind of material occurs special interaction with GLUT1 and provides scientific basis.
On this basis, new method is used for screening 22 compounds as shown in Figure 4, and the predominantly flavones in radix scutellariae carries
Thing, streptomyces metabolite and bacillus metabolite are taken, these compounds is have studied respectively and sieves capillary color in post
Compose post, the cells of normal HEK 293 overexpress the cells of HEK 293 as phase as capillary chromatographic column, the GLUT1 of interaction phase
Chromatographic behavior in the capillary chromatographic column of interaction phase.It was observed that FSB-4, FSB-6, FSB-7, FSB-8, SL-1, SL-2,
SL-5, SL-6, SL-7, STR-1~7 chromatographic peak profile in three kinds of capillary chromatographic columns are held essentially constant, and DMSO peak
Deformationization is consistent, belongs to negative compound, as shown pairs of the representative FSB-4 therein in three kinds of capillaries in Fig. 3 (B)
Compare peak shape；On the contrary, FSB-1, FSB-2, FSB-3, FSB-5, SL-3, SL-4 in three kinds of capillary chromatographic columns peak shape change with
EGCG is similar, as shown contrast peak shapes of the wherein representative FSB-1 in three kinds of capillaries in Fig. 3 (B), it is seen that compound exists
Overexpress in capillary of the GLUT1 cells as interaction phase, retention time postpones and peak height reduces, hangover is serious, judges
They exist with GLUT1 specifically binds, and belongs to positive compound.We are screened to 22 compounds, and pass through NLC
The kinetic parameter of the theoretical reactive compound to filtering out is calculated.To this, we have equally been carried out pair using ICCE methods
Than research, as a result show (Fig. 5), the Capillary Electrophoresis behavior of 22 compounds obtained by two methods is consistent, i.e., with GLUT1 whether
It is consistent in the presence of the selection result of interaction, and the kinetic parameter uniformity of gained positive compound is good.
Glucose competitive binding experiment：In order to whether be accounted for when verifying that the compound with binding activity is combined with GLUT1
According to be avtive spot that glucose molecule is combined with GLUT1.This experimental design EGCG, binding activity compound FSB-1,
FSB-2, FSB-3, FSB-5, SL-3, SL-4 target GLUT1 glucose competitive binding experiment, use overexpression GLUT1's
HEK293 cells carry out glucose competitiveness HPCE experiments as the capillary of interaction phase.Using GLUT1 as target spot, buffering
The glucose of different quality is added in liquid respectively, it is molten to be configured to the 40mM PB bufferings containing 0mM, 1mM, 5mM and 10mM glucose
Liquid, the positive compound of sample introduction same concentrations respectively under four kinds of buffer conditions.Positive compound is contrasted in various concentrations grape
Electrophoresis behavior in sugared buffer solution, judge that they whether there is competitive binding between glucose, so as to judge they with
Whether GLUT1 occupies the binding site of glucose when combining.Wherein represent EGCG and the result of glucose competitive assay is shown in Fig. 3
(C), show that peak height rise, peak shape is by having with reference to special as the increase that concentration of glucose is added in buffer solution, EGCG gradually discharge
The hangover broadening peak shape of point is changed into without the narrow and sharp peak shape with reference to feature.This absolutely proves EGCG and glucose competitive binding
What the avtive spot on GLUT1, i.e. EGCG occupied when being combined with GLUT1 is glucose binding site.It is similar with its, FSB-1 and
FSB-3 also shows with concentration of glucose increase is added in buffer solution and peak height rise occurs, the phenomenon of compound release.With this
On the contrary, increasing with concentration of glucose is added in buffer solution, FSB-2 and FSB-5, SL-3, SL-4 peak height, peak shape be not obvious
Change, shows different from glucose combination GLUT1 site, and wherein representative compound FSB-5 result is shown in Fig. 3 (C).To this
We have carried out competion experiment comparative study again by ICCE methods, reactive compound obtained by two methods of display with
Whether GLUT1 occupies glucose site when combining is consistent.
Embodiment 3, tumour cell as interaction phase capillary chromatographic column to the sieve of part binding activity compound
Choosing
Preparation method and GLUT1 overexpression of the A549 tumour cells as the capillary chromatographic column of interaction phase
HEK293 cells are identical as the preparation method of the capillary chromatographic column of interaction phase.This patent is made by A549 tumour cells
For the capillary chromatographic column for the phase that interacts, demonstrate DMSO, EGCG, FSB-1 and FSB-2 and A549 tumour cells whether there is
Interaction.Such as Fig. 6 it can be found that when A549 tumour cells are as interaction phase, DMSO peak height peak shapes do not have notable area
Not, electrophoresis shows typical negative compound.And EGCG, FSB-1 and FSB-2 show as the characteristics of positive compound,
Retention time postpones and peak height reduces, peak shape substantially reduces hangover, and the cells of HEK 293 with GLUT1 overexpressions are as phase interaction
It is consistent with the result of phase, while illustrate the high expression of GLUT1 acceptors of A549 cell surfaces.
Capillary chromatography of the other biological materials such as embodiment 4, in vitro lung tissue or tumor tissues as interaction phase
The preparation of post and the screening to amount of activated compound
The drug screening new method established of the present invention, interaction can be not only mutually cell (including overexpression GLUT1
Cell, tumour cell etc.), can also be the other biological material such as in vitro tissue or tumor tissues.By mouse normal lung tissue,
After the tumor tissues for the heterograft tumour that people source A549 lung carcinoma cells are established are peeled off, 12- is fixed with 4% paraformaldehyde lucifuge
After 48h, it is placed in 75% ethanol, it is stand-by is stored in 4 DEG C of refrigerators.During experiment, by the in vitro tissue or tumour after above-mentioned fixation
In tissue injection post sieve capillary, length 0.5-5mm, rinsed with 40mMpH 7.4 phosphate buffer standby after balancing.
Fig. 7 be in vitro lung tissue (b) and tumor tissues (c) as interaction phase when HPCE scheme.As a result DMSO is shown
On post sieve capillary, Isolated-lung setup action interaction phase, capillary chromatographic column of the tumor tissues as interaction phase,
Because in vitro lung tissue and tumor tissues use form stable, the pressure of system is added to a certain extent, so during appearance
Between postpone.But other electrophoresis behaviors are basically identical, peak height peak shape does not have significant difference, shows typical negative compound
Feature.And by being contrasted to the peak shape of EGCG, FSB-1, FSB-2 in three kinds of capillary chromatographic columns, it is seen that these compounds exist
Peak shape shows certain broadening on the capillary chromatographic column of Isolated-lung setup action interaction phase, exists certain non-specific
Property interaction；And on capillary of the tumor tissues as interaction phase compared with vitro lung tissue, retention time delay
And peak height reduction is more obvious, hangover is serious, judges that they have specificity between tumor tissues and interacted, furtherly
High expression of the bright GLUT1 in tumor tissues.Because ICCE methods can not be surveyed to the combination of compound and in vitro tissue
Fixed, this part is not verified by ICCE methods, more illustrates that this method has widened application, by different biomaterials
Capillary column is introduced as interaction and is mutually used to study intermolecular interaction, with more actual application value, Ke Yitong
Cross the interaction of research people's source tumor tissues and targeted drug, there is provided the optimal treatment targeted drug of binding kineticses parameter,
Laid the foundation for clinical treatment tumour preferred therapeutic targeted drug, there is provided completely new approach and scientific basis.
Embodiment 5, cellular level and the horizontal antitumor activity experiment of animal
The strong positive compound obtained for HPCE preliminary screenings, this experiment is to EGCG, FSB-1, FSB-2, FSB-3, SL-
3rd, SL-4 choose respectively 60 μM of concentration points, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, using mtt assay study compound pair
The effect of A549 growth of tumour cell.The light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD490nm, to various concentrations
Medicine group carries out t- inspections with not dosing group, have the then explanation of significant difference under corresponding concentration the medicine to A549 cells
It is inhibited.Meanwhile the average inhibition under each concentration is calculated, inhibiting rate-drug concentration profile is drawn, sees Fig. 8.This
Experimental result is that above experimental conditions count to obtain three times according to operation repetitive.As a result show that 100 μM of EGCG have with not dosing group
There were significant differences, and other concentration groups do not have significant difference then；20 μM, 40 μM, 60 μM, 80 μM, 100 μM of FSB-1 and SL-4
All there is significant difference with not dosing group；80 μM, 100 μM FSB-2, FSB-3 and non-dosing group all have significant difference, other
Concentration is then not significantly different；The SL-3 of each concentration and non-dosing group all do not have significant difference.With Excel linear sides
Journey, calculate the IC of compound50Value, is shown in Fig. 5.The IC of compound50It is worth smaller, shows that the inhibitory action of medicine is stronger.Thus may be used
See, the screening at molecular level result and cellular level antitumor activity experimental result of compound are basically identical.
The strong positive compound and anti tumor activity in vitro experimental result obtained for HPCE preliminary screenings, study chemical combination
The effect of thing EGCG, FSB-1 and FSB-2 to Non-small cell lung carcinoma cell (A549) tumour growth in Mice Body.From in
The Balb/nu-nu mouse of the A549 of exponential phase and 6~8 week old build nude mice lotus knurl model, including physiological saline group conduct
Blank control group, administration EGCG (80mg/kg) are used as positive controls, have studied FSB-1 groups (80mg/kg) and FSB-2 groups
(80mg/kg) carries out pharmacodynamic evaluation for tumor model.Weighed within the 1st day nude mice and the oxter injection on the right side of nude mice in experiment
A549 cells (5 × 106Individual/only), (tumour reaches about 35mm after injection cell 10d3), nude mice is only randomly divided into according to every group 6
4 groups, start to be administered.It is administered once within every three days, carrying out body weight to nude mice before administration every time weighs and knurl cubing.9 are administered altogether
It is secondary, mouse was put to death in the 27th day after administration, peel off tumour, claim knurl weight.Calculate the average value and tumor suppression of every group of knurl weight
Rate, tumor control rate=(the average knurl weight of the average knurl weight-experimental group of blank group) average knurl weight × 100% of/blank group.Fig. 9 (A~
E) show, compared with physiological saline group, EGCG, FSB-1 and FSB-2 there are significant difference (* * * P to tumor suppression situation<
0.001,**P<0.01,*P<0.05), tumour inhibiting rate is respectively：71.51%, 64.69%and 61.73%.FSB-1 and FSB-2
On mouse weight influence, there was no significant difference, illustrates that FSB-1 and FSB-2 toxicity is smaller；But EGCG groups on mouse weight influence with
For physiological saline group compared to there is significant difference, this prompting EGCG may have certain side effect.
Some specific embodiments are described in detail herein, but this is intended only as saying goal of the invention example
It is bright, the scope without limiting following claims.It should be appreciated that different substitutions to concrete scheme described herein, change and
Modification is all without departing from the connotation and extension defined in the claims in the present invention, so as to belong to the application hair claimed
Bright scope.
Claims (15)
1. a kind of capillary, its phase of sieving and interact with post, wherein the aperture of post sieve is controllable, available for intercepting conduct
The biomaterial for the phase that interacts.
2. capillary according to claim 1, wherein post sieve may be provided at the middle part or end of the capillary
Portion, or post sieve are connected to the end of the capillary.
3. capillary according to claim 1, wherein the biomaterial include cell, mitochondria, tissues in vitro,
Tumor tissues, carrier band biological target solid matter.
4. capillary according to claim 3, wherein the cell is that the cell that large biological molecule overexpresses or tumour are thin
Born of the same parents, the high expression of macromolecular acceptor of cell surface, and keep native conformation and bioactivity.
5. capillary according to claim 1, wherein the capillary is vitreous silica capillary.
6. a kind of method for preparing capillary any one of claim 1-5, comprises the following steps：
Pre-treatment is carried out to capillary；
Post sieve material is pre-processed and forms post sieve, the post sieve is arranged on the middle part or end of the capillary
Portion, or the end for making the post sieve be connected to the capillary, so as to form the capillary with post sieve；Wherein, it is if described
Post sieve is arranged on the end of the capillary or is connected to the end of capillary, then needs capillary end with having detection window
The end of the capillary of mouth is attached by connector；
To being cultivated or being handled and is fixed as the biomaterial of interaction phase；And by the biology after fixation
In the material injection capillary with post sieve so that the biomaterial is intercepted in post sieve rear end as interaction
Phase.
7. according to the method for claim 6, wherein the pre-treatment step is included capillary successively with methanol, ultrapure
Water, 0.1M HCl, ultra-pure water, 0.1M NaOH, ultra-pure water are rinsed.
8. according to the method for claim 6, wherein the external diameter of the internal diameter of the connector and the capillary matches.
9. according to the method for claim 6, wherein the fixing step to biomaterial is included to the biomaterial
Handled using 4% paraformaldehyde.
10. a kind of capillary electrophoresis system, including：
Capillary any one of claim 1-5；
For by the device of capillary described in biomaterial or Sample introduction；
The device of electrophoretic procedures is carried out to the sample for importing the capillary；And
The equipment detected to the sample.
11. a kind of method of Capillary Electrophoresis, including：
Capillary any one of claim 1-5 is provided；
Electrophoretic apparatus is provided；
By capillary described in biomaterial or Sample introduction；
Electrophoretic analysis is carried out to the sample；And
The sample is tested and analyzed.
12. capillary any one of a kind of claim 1-5 is in the purposes of biological technical field, including described in use
Capillary carries out electrophoretic analysis.
13. purposes according to claim 12, wherein the biotechnology includes drug screening, large biological molecule and active ligand
Interaction is purified and identified with compound phase interaction, drug ingedient analysis, active constituents of medicine.
14. purposes according to claim 12, wherein the electrophoretic analysis is Capillary Electrophoresis.
15. purposes according to claim 14, wherein the Capillary Electrophoresis is capillary array electrophoresis or chip capillary electricity
Swimming.
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