CN110201419A - It is a kind of using polyvinyl alcohol microparticles as membrane flexibility column of carrier and preparation method thereof - Google Patents
It is a kind of using polyvinyl alcohol microparticles as membrane flexibility column of carrier and preparation method thereof Download PDFInfo
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- CN110201419A CN110201419A CN201910385950.3A CN201910385950A CN110201419A CN 110201419 A CN110201419 A CN 110201419A CN 201910385950 A CN201910385950 A CN 201910385950A CN 110201419 A CN110201419 A CN 110201419A
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- polyvinyl alcohol
- membrane
- cell membrane
- carrier
- alcohol microparticles
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- 229920002451 polyvinyl alcohol Polymers 0.000 title claims abstract description 115
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- 238000002360 preparation method Methods 0.000 title claims abstract description 19
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- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 14
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Classifications
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- B01D15/08—Selective adsorption, e.g. chromatography
- B01D15/10—Selective adsorption, e.g. chromatography characterised by constructional or operational features
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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
- B01D15/22—Selective adsorption, e.g. chromatography characterised by constructional or operational features relating to the construction of the column
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01J2220/00—Aspects relating to sorbent materials
- B01J2220/40—Aspects relating to the composition of sorbent or filter aid materials
- B01J2220/48—Sorbents characterised by the starting material used for their preparation
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- B01J2220/4868—Cells, spores, bacteria
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/889—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 monitoring the quality of the stationary phase; column performance
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Abstract
The invention discloses a kind of using polyvinyl alcohol microparticles as membrane flexibility column of carrier and preparation method thereof, belongs to membrane flexibility column technology field.The membrane flexibility column is cell membrane stationary phase to be made with cell membrane package polyvinyl alcohol microparticles, then cell membrane stationary phase is made using wet method dress post.Cell membrane stationary phase is made with cell membrane package polyvinyl alcohol microparticles, since polyvinylalcohol microsphere ball surface has hydroxyl abundant and aldehyde radical, not only can with cell membrane by hydrophobic effect in conjunction with, aldehyde groups can also be by reacting with phosphatide amino group abundant on film, to achieve the purpose that allow polyvinyl alcohol microparticles and cell membrane to be covalently attached, keep fixation of the cell membrane in stationary phase more stable, to extend the service life of membrane flexibility column.It is easy to fall off to improve cell membrane when silica gel is carrier to a certain extent simultaneously, the problem of pH narrow application range.
Description
Technical field
The invention belongs to membrane flexibility column technology fields, and in particular to a kind of using polyvinyl alcohol microparticles as the cell of carrier
Membrane chromatography column and preparation method thereof.
Background technique
Membrane flexibility is a kind of new technology that identification target components are effectively directly screened from complex system.Cell membrane
Chromatography is that the cell membrane of active mass or cell is fixed on Silica Surface, is prepared into cell membrane stationary phase, is filled using wet process
Membrane flexibility column is made in column.Using buffer solution as mobile phase, drug is solute or addition in mobile phase, uses liquid chromatography
The interaction of research drug and cell membrane and receptor in stationary phase in a dynamic condition.
Chromatograph packing material is the core material that liquid chromatogram reaches component separation, directly influences the separative efficiency and inspection of chromatography
The accuracy of measured data.Therefore the research and development of new chromatographic filler are always the hot spot of chromatography scholar concern.Present chromatography
Filler can be divided into three categories according to its host material: inorganic matrix filler, organic substrate filler and composite material.Wherein silica gel
Matrix chromatogram packing is current using the most due to having many advantages, such as that mechanical strength is big, column effect is high, packing material size and aperture are controllable
Extensive one kind inorganic filler.But such chromatograph packing material has following two obvious shortcoming: first is that pH use scope is narrow, in strong acid or
Strong base solution will lead to silica gel dissolution, be restricted its application；Second is that the remaining silicone hydroxyl of Silica Surface has adsorption activity,
The hangover of bee shape especially is shown as to the separation of alkali compounds, influences separating effect, limits its further application.
High molecular polymer matrix has many advantages, such as good bio-compatibility, chemical stability and is easy to derivatization.
Polyvinyl alcohol (Polyvinyl alcohol, abbreviation PVA) is a kind of quite extensive water soluble polymer material of purposes
Material, is generated by polyvinyl acetate through alkali catalyzed alcoholysis, and relative molecular mass is 20000~200000.The object of polyvinyl alcohol
Rationality matter is mainly determined that it is also different that degree of polymerization difference will lead to molecular weight by molecular weight and alcoholysis degree.Polyvinyl alcohol is due to molecule
In containing compared with polyhydroxy and have good water solubility；Molecule aggregation and there is good film forming, cohesion, emulsibility and good
The performances such as good grease resistance and solvent resistant.Therefore it is widely used as biomedical material, high molecular material etc..But it closes at present
It is also rarely reported in by technologies such as membrane flexibility columns of carrier and preparation method thereof of polyvinyl alcohol microparticles.
Summary of the invention
In order to overcome the disadvantages of the above prior art, it is to carry that the purpose of the present invention is to provide one kind with polyvinyl alcohol microparticles
Membrane flexibility column of body and preparation method thereof, it is easily de- which can be effectively improved cell membrane when silica gel is carrier
It falls, the short problem of membrane flexibility column life.
In order to achieve the above object, the present invention is achieved by the following scheme:
The invention discloses a kind of using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, the membrane flexibility column be with
Cell membrane stationary phase is made in cell membrane package polyvinyl alcohol microparticles, then cell membrane stationary phase is made using wet method dress post.
Preferably, the polyvinyl alcohol microparticles are using polyvinyl alcohol as raw material, with glutaraldehyde as cross linker, in acid condition
Under catalysis, it is made using anti-phase suspension-chemical crosslink technique.
It is further preferred that can be adjusted by concentration, glutaraldehyde dosage and the acid catalysed conditions of control polyvinyl alcohol poly-
Rigidity, partial size and the dispersibility of vinyl alcohol microballoon.
Preferably, the average grain diameter of polyvinyl alcohol microparticles is 4 μm.
Preferably, cell membrane uses EGFR cell membrane.
The invention discloses a kind of using polyvinyl alcohol microparticles as the preparation method of the membrane flexibility column of carrier, including following
Step:
1) using polyvinyl alcohol as raw material, with glutaraldehyde as cross linker, under acid condition catalysis, using anti-phase suspension-change
The method for learning crosslinking, is made that spherical form is regular and the polyvinyl alcohol microparticles of size uniformity；
2) cell is cultivated, when cell count is not less than 107When a, culture medium is removed, cell is obtained, isolates cell membrane；
3) cell membrane is configured to cell membrane suspension, cell membrane suspension is added made from step 1) gathers under vacuum conditions
It in vinyl alcohol microballoon, stands overnight after mixing evenly, obtains the cell membrane stationary phase using polyvinyl alcohol microparticles as carrier；
4) it uses wet method dress post to obtain the cell membrane stationary phase that polyvinyl alcohol microparticles are carrier with polyvinyl alcohol microparticles to be
The membrane flexibility column of carrier.
Preferably, in step 1), the average grain diameter of polyvinyl alcohol microparticles obtained is 4 μm.
Preferably, in step 2), the cell of acquisition is suspended to be placed in cell Ultrasonic Cell Disruptor with Tris-HCl to be carried out
It is broken, cell membrane is then isolated by differential centrifugation.
Compared with prior art, the invention has the following advantages:
It is disclosed by the invention using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, with cell membrane wrap up polyvinylalcohol microsphere
Cell membrane stationary phase is made in ball, not only can be with cell membrane since polyvinylalcohol microsphere ball surface has hydroxyl abundant and aldehyde radical
Combined by hydrophobic effect, aldehyde groups can also by being reacted with phosphatide amino group abundant on film, thus reach allow it is poly-
The purpose that vinyl alcohol microballoon and cell membrane are covalently attached, keeps fixation of the cell membrane in stationary phase more stable, to extend thin
The service life of after birth chromatographic column.It is easy to fall off to improve cell membrane when silica gel is carrier to a certain extent simultaneously, pH narrow application range
The problem of.
Further, polyvinyl alcohol microparticles, with glutaraldehyde as cross linker, are urged in acid condition using polyvinyl alcohol as raw material
Under change, it is made using anti-phase suspension-chemical crosslink technique.Therefore, it is urged by controlling concentration, glutaraldehyde dosage and the acid of polyvinyl alcohol
Change condition can regulate and control the rigidity of the polyvinyl alcohol microparticles of preparation, it is made to meet the requirement of chromatograph packing material.
Detailed description of the invention
Fig. 1 is that polyvinyl alcohol and glutaraldehyde cross-linking react schematic diagram.
Fig. 2 is polyvinyl alcohol microparticles (PVA) stationary phase and using polyvinyl alcohol microparticles as the EGFR cell membrane (EGFR/ of carrier
PVA) the infrared spectrogram of stationary phase.
Fig. 3 is polyvinyl alcohol microparticles (PVA) chromatographic column and using polyvinyl alcohol microparticles as the EGFR cell membrane (EGFR/ of carrier
PVA) the flow velocity back pressure relational graph of chromatographic column.
Gefitinib is using polyvinyl alcohol microparticles as EGFR cell membrane (EGFR/PVA) color of carrier when Fig. 4 is different in flow rate
Compose the chromatogram on column.
Fig. 5 is using polyvinyl alcohol microparticles as the selectivity (AI: Kato of EGFR cell membrane (EGFR/PVA) chromatographic column of carrier
Puli, II: diazepam, III: tamsulosin hydrochloride, IV: Gefitinib).
Specific embodiment
In order to enable those skilled in the art to better understand the solution of the present invention, below in conjunction in the embodiment of the present invention
Attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only
The embodiment of a part of the invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill people
The model that the present invention protects all should belong in member's every other embodiment obtained without making creative work
It encloses.It should be noted that description and claims of this specification and term " first " in above-mentioned attached drawing, " second " etc. are
It is used to distinguish similar objects, without being used to describe a particular order or precedence order.It should be understood that the data used in this way
It is interchangeable under appropriate circumstances, so that the embodiment of the present invention described herein can be in addition to illustrating herein or describing
Sequence other than those is implemented.In addition, term " includes " and " having " and their any deformation, it is intended that covering is not arranged
His includes, for example, the process, method, system, product or equipment for containing a series of steps or units are not necessarily limited to clearly
Those of list step or unit, but may include be not clearly listed or for these process, methods, product or equipment
Intrinsic other step or units.
It is provided by the invention using polyvinyl alcohol microparticles as the membrane flexibility column of carrier referring to Fig. 1, it is to be with polyvinyl alcohol
Raw material is prepared for spherical polyvinyl alcohol microparticles using anti-phase suspension-chemical crosslinking method.The cell of adherent growth passes through
Cell membrane is prepared in digestion, differential centrifugation, is then sufficiently mixed cell membrane and polyvinyl alcohol microparticles, utilizes polyvinylalcohol microsphere
Cell membrane is wrapped in polyvinylalcohol microsphere ball surface by the suction-operated of ball and the mobility of cell membrane, and aldehyde groups can also pass through
It is reacted with phosphatide amino group abundant on film, to achieve the purpose that polyvinyl alcohol microparticles and cell membrane is allowed to be covalently attached.So
Membrane flexibility stationary phase is fitted into chromatographic column by wet method dress post afterwards, is constituted a kind of using polyvinyl alcohol microparticles as the thin of carrier
After birth chromatographic column.The membrane flexibility column improve silica gel be carrier when cell membrane it is easy to fall off, membrane flexibility column life is short, pH
The problems such as narrow application range, provides a kind of new cell membrane support materials.
Specifically, using polyvinyl alcohol microparticles as the preparation method of the membrane flexibility column of carrier, comprising the following steps:
1) polyvinyl alcohol microparticles are prepared
Under agitation, 1g surfactant Span-80 is added into 20mL atoleine, constitutes continuous phase oil phase；
After stirring 10min, 5% polyvinyl alcohol water solution of 5mL is added into oily phase, setting revolving speed is 400rpm/min, is added after stirring 3h
Enter 1mL glutaraldehyde as crosslinking agent, the 500 dense HCl of μ L are added immediately as catalyst.Setting revolving speed is 400rpm/min, stirring
2h.After cross-linking reaction, 30min is stood.A small amount of dehydrated alcohol is added, put into a centrifuge and be centrifuged (12000rpm,
20min), supernatant is taken out, sediment is used into dehydrated alcohol, isopropanol and pure water repeatedly, uses G5 sinter funnel mistake
Microballoon is filtered, the polyvinyl alcohol microparticles of size more uniform (4 μm) are obtained.Drying in air dry oven is finally putting into for 24 hours, to obtain white
Color powder is polyvinyl alcohol microparticles.
2) preparation is using polyvinyl alcohol microparticles as the membrane flexibility stationary phase of carrier
The cell count of culture is not less than (107It is a), using 0.25% trypsin digestion, under the conditions of 4 DEG C of 1000g
It is centrifuged 10min, removes culture medium taking precipitate, the PBS buffer solution that 10mmol/L is added is suspended again and in 4 DEG C of conditions of 1000g
Lower centrifugation 10min draws the remaining culture medium of cell surface, is repeated 3 times, cultured cell is separated.Then 5mL is used
The Tris-HCl suspension cell of 50mmol/L is placed in clasmatosis in cell Ultrasonic Cell Disruptor, is centrifuged under the conditions of 4 DEG C of 1000g
10min takes supernatant to be placed in centrifuge tube and is centrifuged 10min under the conditions of 4 DEG C of 12000g, and precipitating is cell membrane, utilizes 10mmol/L
PBS clean cell membrane 1 time.Cell membrane suspension is added in 0.05g polyvinyl alcohol microparticles under the conditions of vacuum is shaken again, is set
In stirring 30min in 4 DEG C of conditions on magnetic stirring apparatus, guarantee to stand overnight after cell membrane is mixed well with polyvinyl alcohol microparticles,
Using the mobility of cell membrane and the suction-operated of polyvinyl alcohol microparticles, wrap up cell membrane sufficiently with polyvinyl alcohol microparticles, i.e.,
It is able to the membrane flexibility stationary phase that polyvinyl alcohol microparticles are carrier.
3) foundation of membrane flexibility column
RPL-ZD10 packing column machine wet process is utilized by the membrane flexibility stationary phase of carrier of polyvinyl alcohol microparticles by what is obtained
It is fitted into 10mm (L) × 2.0mm (I.D.) column core to get using polyvinyl alcohol microparticles as the membrane flexibility column of carrier.
Below with reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
By taking EGF-R ELISA (Epidermal growth factor receptor, EGFR) as an example, EGFR is
It is a kind of be distributed widely in mammalian epithelial cell, Deiter's cells, fibroblast and keratinocyte surface tool
There is the N connection glycoprotein of tyrosine kinase activity.The expression of EGFR and the generation of tumour, proliferation, transfer and angiogenesis are close
Correlation, therefore the signal transduction process that EGFR is mediated has become the novel targets that anti-tumor drug is studied.Cellular membrane chromatography can be from
Target components are filtered out in complex system, with the double action that chromatographic isolation and receptor are affine, are expressed using EGFR receptor height
HEK293 cell, prepare membrane flexibility stationary phase by carrier of polyvinyl alcohol microparticles, can filter out act on EGFR by
The specific component of body.
1, using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, the preparation method is as follows:
1) preparation of polyvinyl alcohol microparticles
Under agitation, 1g surfactant Span-80 is added into 20mL atoleine, constitutes continuous phase oil phase；
After stirring 10min, 5mL 5%PVA aqueous solution is added into oily phase, setting revolving speed is 400rpm/min, and 1mL is added after stirring 3h
The 500 dense HCl of μ L are added as catalyst as crosslinking agent in glutaraldehyde immediately.Setting revolving speed is 400rpm/min, stirs 2h.It hands over
Connection after reaction, stands 30min.A small amount of dehydrated alcohol is added, put into a centrifuge and be centrifuged (12000rpm,
20min), supernatant is taken out, sediment is used into dehydrated alcohol, isopropanol and pure water repeatedly, uses G5 sinter funnel mistake
Microballoon is filtered, the more uniform polyvinyl alcohol microparticles of size are obtained.It is finally putting into drying in air dry oven and for 24 hours, obtains white powder
End is polyvinyl alcohol microparticles.
2) preparation of EGFR cell membrane stationary phase
The EGFR cell for having sticked bottle wall is chosen, to cell count, when cell number is not less than 107When, with 0.25% pancreas
The digestion of protease digestion liquid, obtains cell suspension.Cell suspension is centrifuged 10min in 4 DEG C, 1000rpm, removes the culture of supernatant
Liquid, precipitating are EGFR cell.It is added after physiological saline is suspended again and is centrifuged again in above-mentioned condition, wash away cell surface residual
Culture medium, abandon supernatant, physiological saline cleaning process is repeated 2 times.The Tris- of 5mL pre-cooling is added into obtained cell precipitation
HCl hypotonic buffer solution is placed in ice-bath ultrasonic 30min smudge cells in Ultrasound Instrument, pay attention to being guaranteed at when ultrasound 4 DEG C with
Under, to guarantee the activity of cell membrane.After cell is completed in ultrasonication, with cell crushing instrument rupture of membranes, work 3s, interval 1s, repeats 6
It is secondary, Program reset.1000g is centrifuged 10min under the conditions of 4 DEG C, and cell membrane is suspended in supernatant at this time, and Aspirate supernatant is in addition
In one centrifuge tube, 12000g is centrifuged 20min under the conditions of 4 DEG C, and obtained precipitating is cell membrane, by precipitating physiological saline weight
It is outstanding, and 12000g is centrifuged 20min under the conditions of 4 DEG C again, supernatant is abandoned, by the physiology salt of obtained membrane pellet 5mL
Water is suspended, and sucks 5mL syringe, under the conditions of vacuum is vortexed concussion, mixes, is placed on magnetic stirring apparatus with polyvinyl alcohol microparticles
It stands overnight after 4 DEG C of conditions stir 30min to get using polyvinyl alcohol microparticles as the EGFR cell membrane stationary phase of carrier.
3) using polyvinyl alcohol microparticles as the foundation of the EGFR membrane flexibility column of carrier
Cell membrane stationary phase suspension is vortexed and is mixed, 10mL centrifuge tube is transferred to, 1000g is centrifuged 5min under the conditions of 4 DEG C,
Abandon supernatant, precipitating, which is added 5mL physiological saline and is vortexed, to be mixed, and is repeated aforesaid operations 2 times, is removed and extra is not wrapped in polyethylene
After cell membrane on alcohol microballoon, about 5mL physiological saline vortex is added into precipitating and mixes, is subsequently poured into flushed in advance
In packing column machine, mobile phase is ultrapure water, flow velocity 0.5mL/min, and pressure when filling column is no more than 10MPa, fills the column time
Cell membrane stationary phase can be packed into column core, the membrane flexibility column core filled is fitted into stainless steel column sleeve, is filled by 20min
Enter in liquid chromatograph and use, or is placed in tri-distilled water and is put into 4 DEG C of refrigerators and saves backup.
2, to the characterization of the above-mentioned EGFR cell membrane stationary phase being prepared
The polyvinyl alcohol microparticles for taking dry in right amount polyvinyl alcohol microparticles and cell membrane to wrap up are milled respectively with KBr after mixing
It is dry, use Fourier infrared spectrograph (FT-IR) to be tested in wave-length coverage for 4 000~500cm-1.By sample before test
Product are sufficiently dry, and keep clean.Infrared spectrum is measured, testing result is as shown in Fig. 2, 2955.67cm in figure-1Place corresponds to thin
The end CH of lipid in after birth3With the CH in protein3, illustrate that cell membrane successfully wraps up and polyvinylalcohol microsphere ball surface.
3, it investigates using polyvinyl alcohol microparticles as the permeability of the EGFR membrane flexibility column of carrier
The permeability of chromatographic column is an important parameter in its practical application, the mechanical performance and institute's packing chromatography of microballoon
The permeability of column can be characterized by flow velocity-back pressure relationship of packed column.By wet method dress post by polyvinyl alcohol microparticles and carefully
The polyvinyl alcohol microparticles of after birth cladding are filled in respectively in 10mm (L) × 2.0mm (I.D.) stainless steel column, using water as mobile phase
Its column pressure under different in flow rate is investigated, as a result as shown in Figure 3.Polyvinyl alcohol microparticles chromatographic column and with polyvinyl alcohol microparticles it is
For the EGFR membrane flexibility column of carrier in 0.05~0.30mL/min flow rates, flow velocity back pressure keeps good linear pass
System, and there is not the phenomenon that column bed collapses, show polyvinyl alcohol microparticles chromatographic column and using polyvinyl alcohol microparticles as carrier
EGFR membrane flexibility column has good permeability and connectivity, and mechanical strength with higher has in high pressure, high flow rate
Lower isolated ability, is conducive to screen active constituent from traditional Chinese medicine complex system.
4, it investigates using polyvinyl alcohol microparticles as the EGFR membrane flexibility column flow rate of carrier
It is the EGFR membrane flexibility column of carrier in 0.05~0.30mL/min flow rates using polyvinyl alcohol microparticles, stream
Fast back pressure keeps good linear relationship, when investigation flow velocity is 0.05,0.10,0.15,0.20,0.25,0.30mL/min respectively
The reserved graph of positive drug Gefitinib, as shown in Figure 4.With the increase of flow velocity, the retention time of Gefitinib is shortened, and peak width becomes
It is narrow, but the pressure of chromatographic column also increases with it, therefore selects 0.2mL/min as using polyvinyl alcohol microparticles as the EGFR of carrier
The optimum flow rate of membrane flexibility column.
5, verifying is using polyvinyl alcohol microparticles as the effect of the EGFR membrane flexibility column of carrier
The selectivity of EGFR membrane flexibility column is investigated, and the Gefitinib of successively 5 μ L 0.1mg/mL of sample introduction, 5 μ L are distinguished
The captopril of 0.1mg/mL, the diazepam of 5 μ L 0.1mg/mL, the tamsulosin hydrochloride of 5 μ L 0.1mg/mL, the results showed that sun
Property medicine Gefitinib is withed a hook at the end on EGFR cell membrane, and captopril, diazepam and tamsulosin hydrochloride are as negative control medicine
Object on cell membrane without reserve, as shown in Figure 5.
The result shows that the selectivity of EGFR membrane flexibility column is good.Intercolumniation otherness in the column of EGFR membrane flexibility column
Mainly the retention time using Gefitinib on EGFR membrane flexibility column column is index, and otherness is preparation one in column
EGFR/CMC chromatographic column, is placed in liquid chromatogram, sufficiently after balance, continuously into Gefitinib sample 5 times of 5 μ L0.1mg/mL,
The retention time of the Gefitinib of each sample introduction is recorded respectively.Intercolumniation otherness is preparation 3 simultaneously after the same method
EGFR/CMC chromatographic column is placed in liquid chromatograph after sufficiently balancing using identical chromatographic condition, respectively into 5 μ L 0.1mg/
The Gefitinib sample of mL records the retention time of Gefitinib in every EGFR/CMC chromatographic column respectively, as a result such as 1 institute of table
Show: intercolumniation otherness is good in column.Membrane flexibility column not only have the function of chromatographic isolation and also with have bioactivity,
Therefore the activity time of membrane flexibility column is one of the important indicator of membrane flexibility column.This research and utilization EGFR cell membrane color
The preparation method of column is composed, while preparing 3 EGFR2 membrane flexibility columns, is placed it in liquid chromatogram later, is sufficiently balanced
Afterwards, the ceaselessly Gefitinib sample of 5 μ L 0.1mg/mL of sample introduction, after 72h, Gefitinib is in EGFR membrane flexibility column
On still have apparent reservation, the results are shown in Table 1, and it is preferable living to show that EGFR membrane flexibility column still has after application 72h
Property.
Table 1 is investigated by the reproducibility of the EGFR membrane flexibility column of carrier and activity of polyvinyl alcohol microparticles
In conclusion using polyvinyl alcohol microparticles as the EGFR membrane flexibility column of carrier can be special recognition reaction in
The target components Gefitinib of EGFR cell EGFR receptor, and in the column of EGFR membrane flexibility column and intercolumniation reproducibility it is good,
The activity time of EGFR membrane flexibility column is able to satisfy the requirement of Qualitive test test, therefore using polyvinyl alcohol microparticles as carrier
EGFR membrane flexibility column can be applied to screen retained fraction from traditional Chinese medicine complex system.
The above content is merely illustrative of the invention's technical idea, and this does not limit the scope of protection of the present invention, all to press
According to technical idea proposed by the present invention, any changes made on the basis of the technical scheme each falls within claims of the present invention
Protection scope within.
Claims (8)
1. a kind of using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, which is characterized in that the membrane flexibility column is with thin
Cell membrane stationary phase is made in after birth package polyvinyl alcohol microparticles, then cell membrane stationary phase is made using wet method dress post.
2. according to claim 1 using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, which is characterized in that described poly-
Vinyl alcohol microballoon is using polyvinyl alcohol as raw material, with glutaraldehyde as cross linker, under acid condition catalysis, using anti-phase suspension-
Chemical crosslink technique is made.
3. according to claim 2 using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, which is characterized in that pass through control
Concentration, glutaraldehyde dosage and the acid catalysed conditions of polyvinyl alcohol processed can adjust rigidity, partial size and the dispersion of polyvinyl alcohol microparticles
Property.
4. it is according to any one of claims 1 to 3 using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, it is special
Sign is that the average grain diameter of polyvinyl alcohol microparticles is 4 μm.
5. it is according to any one of claims 1 to 3 using polyvinyl alcohol microparticles as the membrane flexibility column of carrier, it is special
Sign is that cell membrane uses EGFR cell membrane.
6. a kind of using polyvinyl alcohol microparticles as the preparation method of the membrane flexibility column of carrier, which is characterized in that including following step
It is rapid:
1) it using polyvinyl alcohol as raw material, is handed under acid condition catalysis using anti-phase suspension-chemistry with glutaraldehyde as cross linker
The method of connection, is made that spherical form is regular and the polyvinyl alcohol microparticles of size uniformity；
2) cell is cultivated, when cell count is not less than 107When a, culture medium is removed, cell is obtained, isolates cell membrane；
3) cell membrane is configured to cell membrane suspension, polyethylene made from step 1) is added in cell membrane suspension under vacuum conditions
It in alcohol microballoon, stands overnight after mixing evenly, obtains the cell membrane stationary phase using polyvinyl alcohol microparticles as carrier；
4) wet method dress post is used to obtain using polyvinyl alcohol microparticles as carrier the cell membrane stationary phase that polyvinyl alcohol microparticles are carrier
Membrane flexibility column.
7. according to claim 6 using polyvinyl alcohol microparticles as the preparation method of the membrane flexibility column of carrier, feature
It is, in step 1), the average grain diameter of polyvinyl alcohol microparticles obtained is 4 μm.
8. according to claim 6 using polyvinyl alcohol microparticles as the preparation method of the membrane flexibility column of carrier, feature
It is, in step 2), the cell of acquisition is suspended to be placed in cell Ultrasonic Cell Disruptor with Tris-HCl to be crushed, and is then led to
It crosses differential centrifugation and isolates cell membrane.
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