CN107064333A - A kind of membrane flexibility post of utilization APTES modifications and its preparation method and application - Google Patents
A kind of membrane flexibility post of utilization APTES modifications and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to membrane flexibility field, in the silica gel application cell membrane chromatography that specifically a kind of new APTES is modified.There is silica gel after the present invention is handled through APTES higher post to imitate, and greatly reinforce the adhesion of cell membrane and silica gel, column life at least extends to 12 days.Imitate and decline in the most fast preceding 3 day time in post in addition, its reappearance is also brought up within 10% by former 20%.Such improvement is very suitable for rare or is relatively difficult to the biomaterial that obtains, can greatly reduce consumption and the waste of biomaterial.
Description
Technical field
It is a kind of membrane flexibility of utilization APTES modifications specifically the present invention relates to membrane flexibility technical field
Post and its preparation method and application.
Background technology
Cellular membrane chromatography (Cell membrane chromatography, CMC) combines biomaterial and chromatography
Advantage, make it possible to filter out effective active component from complex system.Its principle is very simple, is exactly by cell membrane
It is fixed in stationary phase, by the extraction solution sample introduction of complex system (such as Chinese medicine) into membrane flexibility model, then uses matter
Spectrum or other detectors study the retention behavior of all compounds.The retention behavior of compound is stronger, and it is active component to illustrate it
Possibility it is bigger, it is similar with other affinity chromatography principles.This seminar has successfully established a series of CMC moulds
Type, and successfully built an online complete two-dimentional membrane flexibility screening system.
In general, cell membrane and silica gel are combined by hydrophobic interaction.This adhesion is weaker and not
Stable, this causes cell membrane to be easy to come off from silica gel, so as to cause membrane flexibility column life short, post effect is low.Due to color
Spectrum stationary phase on cell membrane aging and come off, the shortcomings of model has short life, poor repeatability.These shortcomings very great Cheng
Popularization, the application of the model are limited on degree.Especially for some more rare cell categories, its cell concentration it is less or compared with
It is difficult to cultivate, it is more difficult to meet actual screening active ingredients requirement.These problems greatly limit the further genralrlization of the model
Using.The problem of affinity chromatography also once faced similar, and by using covalent bonding mode by proteopexy in fixation
Phase surface, solves the caducous problem of biomaterial.
The content of the invention
In order to solve the above problems, it is applied to present invention employs a kind of silica gel of new APTES modifications in CMC.
APTES ((3-aminopropyl) triethoxysilane), molecular formula is C9H23NO3Si, structural formula is
The present invention by the use of APTES as bridge, be bonded aldehyde radical in Silica Surface, the aldehyde radical can with it is thin
In recent years, the research of tumor stem cell causes the extensive concern of people.They are a kind of with self-renewing energy
The specific tumor cells of power, are played an important role in the Preventive of tumour and migration.Killing tumor stem cell will show
The cure rate for improving tumour is write, while being greatly reduced the recurrence rate of tumour.But as a rule separation and culture Tumor Stem are thin
Born of the same parents are very difficult and time consuming processes.And membrane flexibility long lifespan, the efficiency high, ten of our newly-established APTES modifications
Divide and be applied to tumor stem cell.
Therefore in the present invention, we establish the HepG2 that a kind of new APTES is modified by the APTES silica gel modified
The membrane flexibility (Cancer stem cell membrane chromatography, CSCMC) of tumor stem cell, referred to as
HepG2/CSCMC chromatographic columns.In order to confirm the validity of this new model, We conducted a series of research, including post
Effect, column life and selectivity etc..In the present invention, we select a kind of famous salviamiltiorrhizabung, are used as preferable complex system
Carry out active ingredient screening.Have been reported that research has shown that, between 1981 to 2006 years, clinically 70% newly batch medicament sources in
Natural products.Therefore the potential active component with resisting tumour stem cells is filtered out from Chinese medicine it is one and very promising grinds
Study carefully.In addition, we also intend using comprehensive two dimensional gas chromatography method, further to improve CMC separating degree.Under these conditions, we take
A full two-dimension analysis system of HepG2 tumor stem cell membrane flexibilities through APTES covalent modifications is built, to the work in the red sage root
Property composition is screened.In conjunction with the test of pesticide effectiveness confirmation in later stage, such as cell killing and cell apoptosis assay, this will be one
The individual effective method that target compound is screened from complicated system, especially for some are rare, be relatively difficult to what is obtained
Biomaterial.Result of study shows, tanshinone IIA, and Cryptotanshinone and dihydrotanshinone I these three compositions are in the Chromatography Models
There is stronger retention behavior, follow-up pharmacological evaluation also demonstrates that their reactive compound really with certain drug effect.
The first aspect of the present invention there is provided a kind of preparation method of utilization APTES membrane flexibility posts modified, including with
Lower step：
A) APTES stationary phases are synthesized：The spherome surface of silica is modified with APTES, so that silica gel
By APTES and the bridging of glutaraldehyde on surface, bonding obtains the aldehyde functions of an end dissociative；(C.-E.；
Wiedmer,S.K.；J.-H.；Sakeye,M.；Lokajová,J.；Riekkola,M.-L.Journal of
pharmaceutical and biomedical analysis 2012,71,1-10.)
B) the membrane flexibility post of APTES modifications is prepared：Collect 1 × 107To 5 × 107(preferably 3.5 × 107) cell,
10min is centrifuged under the conditions of 110 × g, is then washed with PBS three times；Then add PBS and cell suspension is made, cell is used at ultrasonic wave
Manage device rupture of membranes；Obtained suspension centrifuges 1000 × g, 10 minutes；Precipitation is abandoned, then supernatant centrifuges 12000 × g 20min；
Obtained precipitation is suspended with 5ml PBS produce membrane suspensions again；Cell membrane stationary phase (CMSP) is true by cell membrane suspension
React and prepare with 0.04g APTES silica gel under empty stirring condition；Above all of program is carried out under the conditions of 4 DEG C；Incubate overnight
Educate after 12h, cleaned three times under the conditions of 1000g, 10 minutes using PBS；Gained precipitation be suspended again with PBS it is floating, with LC pumps with
PBS is that mobile phase carries out wet method dress post；Obtained CMC posts (10mm × 2mm internal diameters) are loaded in 0.2.ml min-110mM acetic acid
The equalized temperature 1h of 37 DEG C of holding under the flow velocity of ammonium, until post pressure and baseline are steady.CMC posts are stored in 4 DEG C of ammonium acetate.
Cell in described membrane flexibility post is HepG2 tumor stem cells or HepG2 tumour cells.
In a preferred embodiment of the invention, the cell in described membrane flexibility post is that HepG2 Tumor Stems are thin
Born of the same parents, described membrane flexibility post abbreviation HepG2/CSCMC chromatographic columns.
It is furthermore preferred that the cell in described membrane flexibility post is third generation HepG2 tumour microspheres.
There is provided the membrane flexibility post that a kind of utilization APTES is modified, described membrane flexibility for the second aspect of the present invention
Silica gel in post is the silica gel after being handled through APTES, i.e., the spherome surface of silica is modified with APTES, made
By APTES and the bridging of glutaraldehyde on Silica Surface, bonding obtains the aldehyde functions of an end dissociative.
It is preferred that, described membrane flexibility post is prepared using above-mentioned preparation method.
In a preferred embodiment of the invention, the cell in described membrane flexibility post is that HepG2 Tumor Stems are thin
Born of the same parents or HepG2 tumour cells.
It is furthermore preferred that the cell in described membrane flexibility post is third generation HepG2 tumour microspheres.
The third aspect of the present invention there is provided a kind of analysis system through the APTES complete two-dimentional HepG2/CSCMC modified, by
The series of high efficiency liquid chromatographic system of Agilent 1200, binary pump system, unit pumping system, insulating box, on-line degassing machine, enter automatically
Sample device, automatically controlled ten-way valve composition, and by Agilent MassHunter work stand controls (Agilent Technologies, Palo
Alto,CA,USA).HepG2/CSCMC chromatographic columns (internal diameter 10 × 2mm, 5 μm) are 10mM vinegar as the first dimension chromatogram, mobile phase
Acid ammonium solution, flow velocity is 0.2ml.min-1.Second dimension chromatographic column is Chromolith Performance RP-18e
Monolithic integral posts (100mm × 4.6mm internal diameters, Merck, Darmstadt, Germany), mobile phase is solvent orange 2 A (0.1% first
Acid) and solvent B (acetonitrile), and gradient elution is carried out with 3.5ml.min-1 flow velocity.Second chromatogram gradient sets as follows：0~
4min, 10%B-30%B；4-9 minutes, 30%B-75%B；9-9.01min, 75%B-10%B；9.01-10min, 10%B.
There is detailed elaboration (Chen, X. in the paper that the carrying out practically scheme of two-dimentional system is delivered before this seminar；Cao,Y.；
Zhang,H.；Zhu,Z.；Liu,M.；Liu,H.；Ding,X.；Hong,Z.；Li,W.；Lv,D.Analytical chemistry
2014,86,4748-4757；Chen,X.；Cao,Y.；Lv,D.；Zhu,Z.；Zhang,J.；Chai,Y.Journal of
Chromatography A 2012,1242,67-74.)。
The membrane flexibility post that the fourth aspect of the present invention is modified there is provided above-mentioned utilization APTES screening active Chinese drug component into
Application in point.
In a preferred embodiment of the invention, described Chinese medicine is the red sage root.
The fifth aspect of the present invention screens active component there is provided a kind of APTES cellular membrane chromatographies modified from Chinese medicine
Method, the cell in described membrane flexibility post is HepG2 tumor stem cells, and described active component is acts on
The active component of HepG2 tumor stem cells；
Comprise the following steps：
A, prepare Chinese medicine extract；
B, analysis system of the structure through the APTES complete two-dimentional HepG2/CSCMC modified, wherein the first dimension chromatographic column is
HepG2/CSCMC chromatographic columns, the second dimension chromatographic column is Chromolith Performance RP-18e monolithic overall
Post, the second dimension Coupled columns have flight time mass spectrum；
C, the Chinese medicine extract described in step A squeezed into described through the APTES complete two-dimentional HepG2/CSCMC's modified
In analysis system, first time separation is carried out using the described first dimension chromatographic column, using the described second dimension chromatographic column to described first
Outflow component in dimension chromatographic column is separated again, using outflow group of the flight time mass spectrum to the Two way chromatograms
Analyzed, obtain flowing out the structural information of component, by constantly switching the complete two-dimentional HepG2/ modified through APTES
Ten direction changeover valves in CSCMC analysis system, obtain a variety of potential active components for acting on HepG2 tumor stem cells
Retention behavior collection of illustrative plates.
In a preferred embodiment of the invention, described Chinese medicine is the red sage root.
It is furthermore preferred that the described active component for acting on HepG2 tumor stem cells be tanshinone IIA, Cryptotanshinone and
Dihydrotanshinone I.
The sixth aspect of the present invention is preparing induction HepG2 there is provided tanshinone IIA, Cryptotanshinone or dihydrotanshinone I
Application in tumor stem cell apoptosis or the medicine of suppression HepG2 tumor stem cell propagation.
The invention has the advantages that：
1st, the present invention have developed a kind of silica gel of new APTES modifications and be applied in membrane flexibility, make cell membrane and
Adhesion between silica gel is greatly enhanced, and mitigates cell membrane aging or obscission, by carrying out surface modification to stationary phase, with
Covalent bond with mode increase the stability that cell membrane is connected with silica gel, so as to further optimize the methodology of CMC technologies, prolong
Grow its column life and improve reappearance.There is silica gel after being handled through APTES higher post to imitate.The processing is greatly reinforced
The adhesion of cell membrane and silica gel, column life at least extends to 12 days.Imitate and decline in the most fast preceding 3 day time in post in addition, its
Reappearance is also brought up within 10% by former 20%.Such improvement is very suitable for rare or is relatively difficult to the biology that obtains
Material, can greatly reduce consumption and the waste of biomaterial.
2nd, the present invention establishes a kind of membrane flexibility of the HepG2 tumor stem cells of new APTES modifications come in screening
Potential active component in the medicine red sage root, shows tanshinone IIA, and Cryptotanshinone and dihydrotanshinone I these three compositions are in the chromatogram
There is stronger retention behavior in model, follow-up pharmacological evaluation also demonstrates that their reactive compound really with certain drug effect.
3rd, to the linguistic term of membrane flexibility model in the present invention, can with the application of the further genralrlization model,
And be highly suitable to be applied for cancer stem cell etc. and be relatively difficult to rare cells obtain or negligible amounts.
Brief description of the drawings
Fig. 1 .APTES modified silica-gels processes and the schematic diagram being covalently attached with cell membrane.
Fig. 2 tumours microspheres as tumor stem cell identification experiment result：(A) HepG2 tumours microsphere surface
The investigation result of CD133 expressions；(B) the investigation result of HepG2 cell surfaces CD133 expressions.
The investigation result of Fig. 3 membrane flexibility posts：(A) the post effect contrast of control group CMC posts and APTES modification group CMC posts
As a result；(B) the long-acting investigation (n=6) of retention times of the positive drug GFT on control group and the CMC posts of APTES modification groups；(C)
The selectivity of HepG2/CSCMC models investigates result (1 is DXM, and 2 be GFT).
Retention behavior of the mixed mark solution of four kinds of standard items of Fig. 4 (A) on 2D HepG2/CSCMC；(B) red sage root extract
Retention behavior of the interior Multiple components on 2D HepG2/CSCMC (1-11 molecular formula are as shown in table 1).
Fig. 5 (A) HepG2-CSCs cell proliferation experiment results；(B) HepG2 cell proliferation experiments result；(C) tanshinone
IIA, Cryptotanshinone, dihydrotanshinone I, the IC of Gefitinib50Be worth in HepG2 (respectively 53.050 μM, 34.140 μM,
3.661 μM and 24.230 μM) and HepG2-CSCs in (be respectively 10.300 μM, 17.850 μM, 2.534 μM and 45.740 μM) enter
Row contrast.(*P<0.05 representative has significant difference with negative control tanshin polyphenolic acid B group,**P<0.01 represents and negative control pellet
Phenolic acid B group has pole significant difference, n=5).
Fig. 6 (A) tanshin polyphenolic acid B, tanshinone IIA, Cryptotanshinone, dihydrotanshinone I, Gefitinib are to HepG2-CSCs
The apoptosis experiment flow cytomery result of (left side) and HepG2 (right side) cell；(B) between HepG2-CSCs and HepG2 cells
Apoptosis and the death rate Statistical Comparison result (**p<There is pole significant difference between 0.01 representative and control group,△△p<0.01
Represent and there is pole significant difference, n=3 between positive drug Gefitinib administration group).
Embodiment
The embodiment provided with reference to embodiment the present invention elaborates.
Embodiment 1
1. material and method
1.1 chemicals and experiment material
Silica gel (5 μm,) Qingdao Mei Gao Chemical Co., Ltd.s (Qingdao, China) are purchased from, using preceding in 120 DEG C of work
Change.Gefitinib (GFT, purity>99.5%) it is positive reference substance, dexamethasone (DXM, purity>99.5%) it is negative control
Product, are to be purchased from Mei Lun Pharmaceuticals Ltds (Dalian, China).Tanshin polyphenolic acid B, tanshinone IIA, Cryptotanshinone and dihydrotanshinone I
(purity>99.5%) it is also to be purchased from Mei Lun Pharmaceuticals Ltds (Dalian, China).The red sage root is available from Leiyunshang Pharmaceutical Co., Ltd.
(Shanghai, China).(3- aminopropyls) triethoxysilane (APTES, purity ＞ 99.5%) and glutaraldehyde (GDD, purity ＞
99.5%) Sigma companies, (Missouri, the U.S.) are come from.DMEM culture mediums：The DMEM culture mediums and phosphate of F-12 additions are slow
Fliud flushing (PBS) is Hyclone Products (Thermo Fisher).Hyclone (FBS), B27, bFGF, EGF, insulin are
It is purchased from gbico Life Sciences Co., Ltd (Australia).Dimethyl sulfoxide (DMSO) (DMSO), penicillin, streptomysin and trypsase
It is to buy (Rockville, MD, USA) from GIBCOBRL companies.Ultra-pure water is by Milli-Q A10 ultrapure water production systems
It is prepared by (Millipore, Bedford, MA, USA).Acetonitrile (Merck, Germany) and formic acid (Merck, German) are HPLC grades.Other
Reagent is analysis level.
1.2 instrument
Electronic balance model AND HA-202M (Japan) used.Centrifuge model HITACHI CR21GIII are purchased from
Hitachi Co., Ltd. (Japan).CCK-8 kits and apoptosis kit be purchased from green skies Co., Ltd (Shanghai,
China).Highly effective liquid phase chromatographic system is provided by Anjelen Sci. ＆ Tech. Inc angilent-1100, (California, it is beautiful
State), and angilent-6200 mass spectrographs are connected with, use Mass Hunter LCMS work stations.Ultrasonic cell disintegration instrument
JY92-IIN is purchased from new sesame biotechnology (Ningbo, China).Automatically controlled 10 port valve (mxp9960-000, Rheodyne, Rohnert
Park, CA, USA), equipped with CMC posts and Chromolith Performance RP-18e integral posts (100mm × 4.6mm internal diameters,
Merck,Darmstadt,Germany)。
The preparation of 1.3 samples and standard liquid
The red sage root is first ground into powder, extracts 80 DEG C 1 hour with 50% alcohol reflux, is then condensed into 10mg/mL.Before analysis
With 0.2 μm of membrane filtration.Gefitinib, dexamethasone, tanshin polyphenolic acid B, tanshinone IIA, the mark of Cryptotanshinone and dihydrotanshinone I
Quasi- solution is to be dissolved in 20mM concentration in DMSO, matching while using.
1.4 cell line
He pG2 are chosen herein as the representative of liver cancer cell lines, from the purchase of Chinese Academy of Sciences's cell bank Shanghai branch.
Cell is cultivated in the culture medium (DMEM) containing 10% hyclone (FBS), is placed in 5%CO2,37 DEG C of incubators.
Studies have reported that proving, tumour microsphere can represent tumor stem cell.In order to obtain HepG2 tumour microspheres,
HepG2 cells are in culture dish with 1 × 104Cells/mL concentration Secondary Cultures.Its culture environment is trained for serum-free DMEM-F12
Base is supported, wherein added with 2% (v/v) B27,10ng/ml bFGF, 20mg/mL EGF and 5mg/ml insulin.Tumour microsphere
1min is digested under the conditions of 37 DEG C with pancreatin within every six days, then with 1 × 104The concentration Secondary Culture of cells/ml.In this research
The cell used is the 5th generation cell in exponential phase.
1.5 animal
All nude mices (female, 18-20 grams, 6-8 week) be purchased from Shanghai Chinese Academy of Sciences Experimental Animal Center (Shanghai, in
State).Mouse is placed under gnotobasis and raised, and adapts to environment and is used after one week.All programs are all according to The 2nd Army Medical College
The guilding principle of the animal committee is carried out, Shanghai, China).
Tumor stem cell in 1.6 identification He pG2 tumour microspheres
Liver-cancer stem cell can be confirmed by a variety of phenotypes of table its surface expression, such as CD133, however, only
Hepatoma liver cells are confirmed by these phenotypes, its sensitivity and specificity is poor.In fact, there is no so far generally acknowledged
Liver stem cells phenotypic markers thing.Generally research thinks that the culture of tumour microsphere is a kind of training of tumor stem cell now
Support separation method.In our current research, culture HepG2 tumour microspheres have the characteristic of liver stem cells, and thin using tumour is included
The experiment of born of the same parents' balling ratio, tumor formation rate experiment and surface marker (CD133+And CD133-) quantitative method identified it.More
Importantly, tumour microsphere manually can be cultivated and passed on, this for we subsequent experimental it is significant.
The balling ratio experimentation of tumour microsphere is as follows, is inoculated with respectively on six different orifice plates first a number of
HepG2 cells and digested from third generation HepG2 tumour balls isolated unicellular.By the serum-free F-12DMEM of 6 days
Nutrient solution culture, the balling-up number to tumour is counted and compares two group differences (n=3).
Determined for tumour Forming ability, respectively by a number of HepG2 cells or from third generation HepG2 tumour balls
Digestion isolated unicellular (in the matrigel for being dispersed in 100 milliliters) is inoculated into mammary gland of mouse fat, then studies each
The formation difference (n=5) of tumour between group.
The surface marker (CD133) of tumour ball is detected by flow cytometer (FCM).In brief, third generation tumour is micro-
Individual cells are separated into after spheroid enzymolysis, PBS is washed twice, is then suspended again with PBS.The mouse for adding 20 μ L PE marks inside resists
People's CD133 antibody, and in dye solution 4 DEG C be incubated 30 minutes.Then the buffer solution for cleaning of cell precooling three times.Most
Afterwards, cell is suspended in the PBS of 500 μ L precoolings, uses FACS Calibur CM (Becton Emily Dickinsons company, the U.S.) instrument
It is measured by BD CellQuest softwares.
1.7 synthesis APTES stationary phases
Its course of reaction is as shown in Figure 1.First, the spherome surface of silica is modified with APTES, so that
Make the bridging by APTES and glutaraldehyde on Silica Surface, bonding obtains the aldehyde functions of an end dissociative.The aldehyde groups can
With by being reacted with the abundant phosphatide amino group on film, so as to reach the purpose for allowing silica gel and film to be covalently attached.
1.8 prepare HepG2-CSCs/CMC posts
With the silica gel modified through APTES, step prepares HepG2-CSCMC posts as described below.In brief, 3.5 are collected
×107Cell, centrifuges 10min under the conditions of 110 × g, is then washed with PBS three times.Then add PBS and cell suspension is made, carefully
Born of the same parents use processor for ultrasonic wave rupture of membranes.Obtained suspension centrifuges 1000 × g, 10 minutes.Precipitation is abandoned, then supernatant is centrifuged
12000×g 20min.Obtained precipitation is suspended with 5ml PBS produce membrane suspensions again.Cell membrane stationary phase (CMSP) is
React and prepare with 0.04g APTES silica gel under the conditions of cell membrane suspension vacuum stirring.Above all of program is in 4 DEG C of conditions
It is lower to carry out.After night incubation 12h, cleaned three times under the conditions of 1000g, 10 minutes using PBS.Gained precipitation is mixed with PBS again
Suspend, wet method dress post is carried out by mobile phase of PBS with LC pumps (Waters 996).Obtained CMC posts are loaded (in 10mm × 2mm
Footpath, is purchased from big Puli company day after day) in 0.2.ml min-1The equalized temperature 1h of 37 DEG C of holding under the flow velocity of 10mM ammonium acetates, directly
It is steady to post pressure and baseline.CMC posts are stored in 4 DEG C of ammonium acetate.
1.9 analyze through the APTES complete two-dimentional HepG2/CSMC modified
The complete two-dimentional HepG2/CSCMC of APTES modifications analysis system is by the series of high efficiency liquid chromatogram system of Agilent 1200
System, binary pump system, unit pumping system, insulating box, on-line degassing machine, automatic sampler, automatically controlled ten-way valve composition, and it is prompt by peace
Human relations MassHunter work stand controls (Agilent Technologies, Palo Alto, CA, USA).HepG2/CSCMC chromatograms
Post (internal diameter 10 × 2mm, 5 μm) is as the first dimension chromatogram, and mobile phase is 10mM ammonium acetate solutions, and flow velocity is 0.2ml.min-1.The
Two way chromatograms post be Chromolith Performance RP-18e monolithic integral posts (100mm × 4.6mm internal diameters,
Merck, Darmstadt, Germany), mobile phase is solvent orange 2 A (0.1% formic acid) and solvent B (acetonitrile), and with 3.5ml.min-1's
Flow velocity carries out gradient elution.Second chromatogram gradient sets as follows：0~4min, 10%B-30%B；4-9 minutes, 30%B-
75%B；9-9.01min, 75%B-10%B；9.01-10min, 10%B.
1.10 cells are bred to be detected with Apoptosis
Using cell proliferation detecting kit CCK-8 (green skies Co., Ltd, Shanghai, China), and open to specifications
Open up cell proliferation experiment.Experiment uses GFT as positive control.After cell attachment culture 24h, the GFT of various concentrations is added,
Tanshin polyphenolic acid B, tanshinone IIA, Cryptotanshinone, dihydrotanshinone I, and it is incubated 48h.Then, 10 μ L CCK-8 solution are added per hole,
Be incubated 1.5h under the conditions of 37 DEG C, and using microplate absorbance reader (Bio-RAD instruments,
USA absorbance) is read at 450nm.
With Annexin V-FITC apoptosis detection kit flow cytometers (BD pharmingenTMCA, USA) determine thin
The apoptosis of born of the same parents.The inoculation 5 × 10 per hole on six orifice plates5Cell, is separately added into 20 μM of GFT, tanshin polyphenolic acid B, Cryptotanshinone, the red sage root
Ketone IIA, is incubated 48h.Then, cell is collected, PBS is washed twice.Then Annexin V/FITC reagents are added.Room temperature dark condition
After lower incubation 10 minutes, cleaning cell and resuspension；Then final concentration of 1mg/L propidium iodide is added.Dye the cell finished
Analysis measure is carried out with FACS Calibur instrument (Becton Dickinson, Mountain View, CA, USA).
2. result and discussion
2.1 using tumour ball as tumor stem cell identification
HepG2 cells and HepG2 tumour microsphere balling ratio experimental results are as shown in table 1；
The internal tumorigenesis rate experimental result of HepG2 cells and HepG2 tumour microspheres is as shown in table 2；
As shown in table 1, compare with the cell separated from HepG2 cells, from third generation HepG2 tumour microballoon body cells
Isolated cell has higher balling-up ability, and this shows that HepG2 tumour microballoon body cells are more easy to again than HepG2 cell
Form microsphere.As shown in table 2, third generation HepG2 tumours microsphere cell concentration quantity is more than or equal to 2 × 103.If needing
The morbidity of the induced tumor of HepG2 cells 100%, cell consumption is greater than or equal to 2 × 105, this is tumour microsphere cell concentration
100 times.In a word, HepG2 tumours microballoon body cell has higher tumour Forming ability than HepG2 cell.According to fluidic cell
The result (Fig. 2A and 2B) of instrument, CD133 is 24.09% in the expression quantity of HepG2 microballoon cell surfaces, on HepG2 cells
Only 4.67%, and the phenotype is considered as most common type in hepatic carcinoma stem cell.Therefore, we are eventually through balling-up
Rate experiment, the experiment of tumorigenesis rate and surface marker CD133 etc. are many-sided to be investigated, final to determine third generation HepG2 tumour microspheres
Can as HepG2 tumor stem cells representative.
Table 1.HepG2 and HepG2 are spheroid balling ratio experimental result (n=3)
Table 2.HepG2 and HepG2 microsphere tumorigenesis rate experimental result (n=3)
The reappearance of 2.2 membrane flexibility posts and the raising in life-span
Membrane flexibility post is generally gradually inactivated in use, causes its reappearance low and the life-span only has 3 days or so.
This hinders CMC popularization and application significantly.In order to solve this problem, the present invention once uses paraformaldehyde in research before
Enter luggage post pre-treatment to pillar, successfully strengthen the reappearance of CMC posts, while by pillar life by 6 days or so.
But this process adds the difficulty of CMC posts preparation, thereby increases and it is possible to which the bioactivity to cell membrane has an impact to a certain extent.Cause
This, in terms of we focus on the modification of stationary phase in the present invention.
It is well known that cell membrane is made up of phosphatide, it provides substantial amounts of amino.In consideration of it, we close
Silica gel is APTES- silica gel after into a kind of modification.Silica Surface after modification contains abundant free aldehyde, and it can be with cell
The amino on film surface combines to form stable acid amides covalent bond.
Due to the more difficult culture of liver-cancer stem cell and acquisition, but the column efficiency of APTES modified silica-gels experiment, column life and steady
Qualitative investigation and investigate and need substantial amounts of cell membrane, thus we using HepG2 cell replacement HepG2-CSCs cells as examining
Model is examined, to evaluate the effect of APTES modified silica-gel systems.According to research before, we select GFT as positive control drug
Thing, and detect its retention time on post.According to 1.0 × 107The consumption of individual cell/post, is used after being handled through APTES respectively
Silica gel and untreated silica gel prepare two groups of HepG2/CMC posts (n=5).As shown in Figure 3 B, retention times of the GFT in APTES groups
About 21min, and there was only 10min in the retention time of control group.This shows that the silica gel after being handled through APTES has higher post
Effect.
We are respectively under identical optimum condition, i.e., using 3.5 × 107Under the conditions of the consumption of individual cell/post, make respectively
Silicon and unprocessed silica gel are prepared for two groups of HepG2/CMC posts after being modified with APTES, and continuously investigate 12 days, daily sample introduction 6 times
(n=5).As shown in Figure 3 C, due to being Covalent bonding together on APTES- silica gel, rather than traditional Hydrogenbond, therefore should
Processing has greatly reinforced the adhesion of cell membrane and silica gel, and column life at least extends to 12 days.Decline in addition in post effect most fast
The preceding 3 day time in, its reappearance is also brought up within 10% by former 20%.
2.3 2D HepG2/CSCMC systematic differences
GFT (epidermal growth factor receptor antagonists) and dexamethasone (steroids compound) are selected, respectively as the positive
With negative control medicine, to confirm the selectivity through the APTES 2D HepG2/CSCMC systems modified.Wherein GFT shows stronger
Retention behavior, reach peak value in 17min or so.But DSMS in CMC models substantially without reserve, 2 minutes or so i.e. reach
Peak value.This shows that the system has good selectivity, as shown in Figure 3 C.
The 2D HepG2/CSCMC systems put up are applied to potential reactive compound in the screening red sage root by us.As schemed
Shown in 4B, tanshinone IIA, Cryptotanshinone, dihydrotanshinone I have retention behavior in 2D HepG2/CSCMC systems, red
Phenolic acid B does not retain then substantially.The accurate mass data and abundant isotope fragment provided in addition by flight time mass spectrum are believed
Breath trial has carried out Preliminary Identification, as a result as shown in table 3.
We are also prepared for containing four typical compositions that (tanshin polyphenolic acid B is negative control, tanshinone IIA, the hidden red sage root in addition
Ketone, dihydrotanshinone I be positive control) mixed standard solution, further to confirm the structure of these four materials.Such as Fig. 4 A institutes
Show, the retention behavior of four kinds of compositions is consistent with expection, and corresponding with the selection result.With reference to the first of time of-flight mass spectrometer before
Qualification result is walked, it is tanshinone IIA, Cryptotanshinone and dihydrotanshinone I finally to determine potential main active.
TOFMS Preliminary Identification result of each composition of the red sage root of table 3. in 2D HepG2/CSCMC systems
Note：A. the composition identified through standard items；B. flat peak is not walked within 30 minutes on the first dimension membrane flexibility.
2.4 cells are bred and Apoptosis detection
2.4.1 cell proliferation experiment
Conventional research shows that tanshinone IIA, Cryptotanshinone, dihydrotanshinone I can suppress proliferation of hepatocellular carcinoma HepG 2 cell line,
But seldom there is data to suggest that they have lethal effect to HepG2-CSCs cells.Therefore, these three obtained changes are screened to investigate
The drug effect of compound, we have carried out cell proliferation test, and its result and our garbled data result are consistent, such as Fig. 5 institutes
Show.Dihydrotanshinone I goes out the lethal effect of most strong dose dependent to HepG2-CSCs cells shows, next to that the hidden red sage root
Ketone, tanshinone IIA.Tanshin polyphenolic acid B is then almost without effect.In addition, we are also used as sun using clinical conventional chemical drug Gefitinib
Property control.IC of the Gefitinib in HepG2-CSCs cells50It is worth and is higher than HepG2 cells (24.23 μM) for 45.74 μM, shows
Gefitinib has declined to tumor stem cell killing ability.This is also consistent with the selection result before us, because GFT exists
APTES modification HepG2/CMC peak times be more than 30min, and APTES modify HepG2/CSCMC on only have
16min.In contrast, the IC of tanshinone IIA, Cryptotanshinone and dihydrotanshinone I50Compare HepG2 in HepG2-CSCs cells
Low in cell, this shows that they have more preferable lethal effect to tumor stem cell.Therefore tanshinone IIA, Cryptotanshinone, dihydro
Tanshinone I is confirmed as the composition potentially with anti-HepG2-CSCs activity, and HepG2-CSCs fragmentation effect is better than
Its fragmentation effect to HepG2 cells.
2.4.2 Apoptosis is detected
In order to further determine that the drug effect of filtered out medicine, and assess its growth inhibition work to HepG2-CSCs cells
Whether with related to Apoptosis, We conducted Apoptosis detection.As shown in Figure 6B, cell and tanshin polyphenolic acid B, tanshinone
IIA, Cryptotanshinone and dihydrotanshinone I, GFT (positive control) are incubated after 48h under 20 μm of concentration, compared with control group, are used
Medicine group is in addition to tanshin polyphenolic acid B group, and percentage of cell apoptosis is all significantly raised；This shows to screen from 2D-HepG2/CSCMC systems
The tanshinone IIA arrived, Cryptotanshinone and dihydrotanshinone I can effectively induce HepG2-CSCs Apoptosis.As shown in Figure 6A, flow
Formula cell instrument testing result shows that the percentage of the apoptotic cell of traditional chemical drug Gefitinib drops to from 50.32% (HepG2)
11.93% (HepG2-CSCs), shows it to the active poor of HepG2-CSCs.However, tanshinone IIA, Cryptotanshinone, dihydro
The apoptosis ratio that Tanshinone I compares HepG2 cells in HepG2-CSCs cells has been lifted, and illustrates it in HepG2-CSCs
With more preferable biological activity.
Cell is bred and the result of study of Apoptosis shows that tanshinone IIA, Cryptotanshinone, dihydrotanshinone I are right
HepG2-CSCs has good inhibiting effect, and it is strong to compare HepG2 cytosis effects.This is that three kinds of compounds are demonstrate,proved first
May be effectively in the tangible growth course for suppressing liver-cancer stem cell.
3. conclusion
Shorter column life and relatively low reappearance greatly hinder CMC popularization and application.Therefore, we pass through to solid
It is fixed mutually to carry out surface modification, the stability that cell membrane is connected with silica gel is increased with the way of by covalent bond, so as to further optimize
The methodology of CMC technologies, extends its column life and improves reappearance.Broadly, we improve column life to 12
My god, and during preceding use in 3 days, reappearance is brought up within 10%, former is reported as 20%.Such improvement is very
Suitable for rare or be relatively difficult to the biomaterial that obtains, because the improvement can greatly reduce consumption and the waste of biomaterial.
This research selection tumor stem cell (CSC) is as object, applied in the improved model, while comprehensive two dimensional gas chromatography technology, we
A HepG2/CSCMC system modified through APTES more effectively, stable has been built, has screened and acts on from salviamiltiorrhizabung
The lateral reactivity composition of HepG2 tumor stem cells.Final screening obtains 3 kinds of lateral reactivity compounds, tanshinone IIA, the hidden red sage root
Ketone and dihydrotanshinone I, its IC50 in HepG2-CSCs is respectively 10.30 μM, 17.85 μM and 2.534 μM.
The improvement strategy has expanded CMC application significantly, and is conducive to it to be further commercialized popularization.And not
Only tumor stem cell can be applied in the improved model in this research, other it is rare or be relatively difficult to obtain cell also may be used
To be applied in the model, and efficiently screen from the complication systems such as Chinese medicine potential active component.
The preferred embodiment to the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (10)
1. a kind of preparation method of the membrane flexibility post of utilization APTES modifications, it is characterised in that comprise the following steps：
A) APTES stationary phases are synthesized：The spherome surface of silica is modified with APTES, makes to lead on Silica Surface
The bridging of APTES and glutaraldehyde is crossed, bonding obtains the aldehyde functions of an end dissociative；
B) the membrane flexibility post of APTES modifications is prepared：Collect 1 × 107To 5 × 107Cell, is centrifuged under the conditions of 110 × g
10min, is then washed three times with PBS；Then add PBS and cell suspension, cell processor for ultrasonic wave rupture of membranes is made；What is obtained is mixed
Suspension centrifuges 1000 × g, 10 minutes；Precipitation is abandoned, then supernatant centrifuges 12000 × g 20min；Obtained precipitation uses 5ml again
PBS suspends and produces membrane suspensions；Cell membrane stationary phase be by cell membrane suspension vacuum stirring under the conditions of with 0.04g APTES
It is prepared by silica gel reaction；Above all of program is carried out under the conditions of 4 DEG C；After night incubation 12h, using PBS in 1000g, 10
Cleaned three times under the conditions of minute；Gained precipitation is suspended with PBS again is floated, and wet method dress post is carried out by mobile phase of PBS with LC pumps；Dress
Obtained CMC posts are filled out in 0.2.ml min-137 DEG C of equalized temperature 1h is kept under the flow velocity of 10mM ammonium acetates, until post pressure and
Baseline is steady.
2. the preparation method of the membrane flexibility post of utilization APTES modifications according to claim 1, it is characterised in that institute
Cell in the membrane flexibility post stated is HepG2 tumor stem cells or HepG2 tumour cells.
3. the preparation method of the membrane flexibility post of utilization APTES modifications according to claim 1, it is characterised in that institute
Cell in the membrane flexibility post stated is third generation HepG2 tumour microspheres.
4. a kind of membrane flexibility post of utilization APTES modification, it is characterised in that the silica gel in described membrane flexibility post is
Silica gel after being handled through APTES, i.e., modified the spherome surface of silica with APTES, makes to lead on Silica Surface
The bridging of APTES and glutaraldehyde is crossed, bonding obtains the aldehyde functions of an end dissociative.
5. the membrane flexibility post of utilization APTES modifications according to claim 4, it is characterised in that described cell membrane
Chromatographic column is prepared using any described preparation methods of claim 1-3.
6. a kind of analysis system through the APTES complete two-dimentional HepG2/CSCMC modified, it is characterised in that be by Agilent 1200
Row highly effective liquid phase chromatographic system, binary pump system, unit pumping system, insulating box, on-line degassing machine, automatic sampler, automatically controlled ten
Port valve is constituted, and by Agilent MassHunter work stand controls；HepG2/CSCMC chromatographic columns are used as the first dimension chromatogram, flowing
It is mutually 10mM ammonium acetate solutions, flow velocity is 0.2ml.min-1；Second dimension chromatographic column is Chromolith Performance RP-
18e monolithic integral posts, mobile phase is the formic acid of solvent orange 2 A 0.1% and solvent B acetonitriles, and with 3.5ml.min-1Flow velocity
Carry out gradient elution；Second chromatogram gradient sets as follows：0~4min, 10%B-30%B；4-9 minutes, 30%B-75%B；
9-9.01min, 75%B-10%B；9.01-10min, 10%B.
7. the membrane flexibility post or one kind of a kind of utilization APTES modifications as described in claim 4 or 5 are such as claim 6 institute
The analysis system through the APTES complete two-dimentional HepG2/CSCMC modified stated, the application in screening active ingredient of Chinese herbs.
8. the method that a kind of cellular membrane chromatography of APTES modification screens active component from Chinese medicine, it is characterised in that including with
Lower step：
A, prepare Chinese medicine extract；
B, analysis system of the structure through the APTES complete two-dimentional HepG2/CSCMC modified, wherein the first dimension chromatographic column is HepG2/
CSCMC chromatographic columns, the second dimension chromatographic column is Chromolith Performance RP-18e monolithic integral posts, described
Second dimension Coupled columns have flight time mass spectrum；
C, the Chinese medicine extract described in step A is squeezed into the complete two-dimentional HepG2/CSCMC modified described in step B through APTES
Analysis system in, first time separation is carried out using the described first dimension chromatographic column, using the described second dimension chromatographic column to described the
Outflow component in one-dimensional chromatographic column is separated again, the outflow using the flight time mass spectrum to the Two way chromatograms
Component is analyzed, and obtains flowing out the structural information of component, by constantly switching the complete two-dimentional HepG2/ modified through APTES
Ten direction changeover valves in CSCMC analysis system, obtain a variety of potential active components for acting on HepG2 tumor stem cells
Retention behavior collection of illustrative plates.
9. the method that the cellular membrane chromatography of APTES modifications according to claim 8 screens active component from Chinese medicine, its
It is characterised by, described Chinese medicine is the red sage root.
10. the method that the cellular membrane chromatography of APTES modifications according to claim 9 screens active component from Chinese medicine,
Characterized in that, the active component that HepG2 tumor stem cells are acted in the described red sage root be tanshinone IIA, Cryptotanshinone and
Dihydrotanshinone I.
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| CN110479205A (en) * | 2019-08-15 | 2019-11-22 | 西安交通大学 | A kind of drug screening magnetic Nano material and preparation method and application based on cell membrane |
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| CN107607646A (en) * | 2017-09-14 | 2018-01-19 | 神威药业集团有限公司 | A kind of platelet cell membrane chromatography |
| CN110479205A (en) * | 2019-08-15 | 2019-11-22 | 西安交通大学 | A kind of drug screening magnetic Nano material and preparation method and application based on cell membrane |
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