CN102146425B - Heparin fragment with activity of inhibiting smooth muscle cell proliferation and preparation method thereof - Google Patents
Heparin fragment with activity of inhibiting smooth muscle cell proliferation and preparation method thereof Download PDFInfo
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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- Y02P20/50—Improvements relating to the production of bulk chemicals
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Abstract
The invention discloses a heparin fragment with activity of inhibiting smooth muscle cell proliferation and a preparation method thereof. The preparation method comprises the following steps of: (1) performing enzymatic hydrolysis on heparin by heparinase to obtain enzymatic hydrolysate of heparin; and (2) separating the heparin fragment with the activity of inhibiting smooth muscle cell proliferation from the enzymatic hydrolysate of heparin by an SPR (Surface Plasmon Resonance) sensing technique. The heparin fragment prepared by the method disclosed by the invention has a function of inhibiting the smooth muscle cell proliferation, when the concentration is lower, the inhibition rate of the heparin fragment on the smooth muscle cells is higher than that of heparin, and compared with heparin, the heparin fragment obtained by the invention has very low anticoagulant activity which is only 1.9% of that of the original heparin. Therefore, the method and the prepared heparin fragment disclosed by the invention can be used clinically, and overcome the defect that heparin causes obvious hemorrhage symptom in the use process, so that practical application of the character of heparin of resisting smooth muscle cell proliferation is possible. Thus, the method and product disclosed by the invention have wide application prospects.
Description
Technical field
The present invention relates to have heparin fragment that suppresses the smooth muscle cell proliferation activity and preparation method thereof.
Background technology
Heparin is the Sulfated important member of a kind of height in the glycosaminoglycan family.After it is synthetic in mastocyte, be transported in the various histoorgans, so it extensively exists in vivo.The molecular weight of heparin strand is up to 60,000-100 in the organism, 000Da, and many heparin chains connect by the peptide section again, form huge proteoglycan molecules.Commercial heparin molecule amount is to be come by above-mentioned glycosaminoglycan degraded, and huge sugar chain many places is in process of production interrupted, and has formed molecular weight ranges product between 5000-25000Da greatly.This shows that heparin is made up of the combination chain of different lengths, interchain is then variant again, has caused the height heterogeneity of heparin component.According to composition and the oligosaccharide sequence analysis to the heparin chain, known overall structure and the composition of heparin now substantially.In the chain mainly with uronic acid residue (L-iduronic acid, IdoA; D-glucuronic acid GlcA) alternately occurs with six osamines (D-glucoamine) form.
Heparin clinical application the earliest occurs as anti-freezing and antithrombotic.Studies show that afterwards that it also had biological activitys such as anti-smooth muscle cell proliferation, anti-inflammatory, antitumor mediator's body immunity function.It can be treated and prevent because of wound simultaneously, asthma, atherosclerosis, hypertension, congestive heart failure, pulmonary apoplexy, the ephritis syndromes, acute glomerulonephritis, the proliferation of smooth muscle that baby's congenital heart trouble etc. causes, prevention angiostenosis, avoid or reduce children's and adult hypertensive pulmonary vascular disease, the heart that prevention and treatment coronary heart disease and myocardial infarction patient underwent coronary " Coronary Artery Bypass " " sacculus expansion art " " mounting bracket art " and cerebral embolism patient cause through " intracranial vessel bypass " proliferation of smooth muscle, cerebrovascular restenosis (restenosis).Therefore heparin has multiple medicinal potentiality.But heparin in use can cause tangible bleeding as the Sulfated polysaccharide of a kind of height, the side effects limit that this is main the clinical application of heparin.
The approach that addresses this problem is to rise in the clear and definite heparin respectively the structure of oligose fragment of anticoagulating active and the fragment structure of other biologic activity.
Up to the present, the anticoagulating active oligose fragment of heparin is unique structure of being illustrated.It is one and contains 3-o-sulfate group pentasaccharides fragment.
The effect of heparin pentasaccharides and antithrombin comprises two steps: at first, non-reducing end three glycosylation sequences and ATIII form the recognition complex of a low affinity in the pentasaccharides sequence, induce the ATIII conformational change to cause the formation of high-affinity mixture.Three glycosylation sequences can activate ATIII fully, and two glycosylation sequences of reducing end are unimportant to activation process, but it can stablize the conformation after the activation.
With the exception of this, the structural domain of other biological function it be unclear that in the structure of heparin.
The SPR sensing technology is based on a kind of surface plasma resonance by name (Surface Plasmon Resonance, a kind of sensing technology that physical optics phenomenon SPR) grows up.Its ultimate principle be utilize the change of material molecule (or the affine binding molecule mixture) quality be attached to special metallic film surface can cause near the variation of local indexes of refraction, this variation is changed into electrical signal output, thereby detect the situation of metallic film surface interaction of molecules in real time.Schedule of operation comprises following step: special biomolecules (acceptor) is fixed in golden film surface by chemistry or physical method, makes detection chip; Feed damping fluid to chip surface, clean and obtain the balance baseline; Feed detected sample (solution that namely contains part) then, the association reaction of detector ligand and acceptor; Feed damping fluid again, the dissociating of the mixture that detection reaction forms; Feed regenerated liquid at last and thoroughly wash away sample to be checked, make chip be regenerated.The advantage of this technology is: the exempt from mark, not damaged of sample detect, and detect in real time and the returnability of molecules detected.
Summary of the invention
An object of the present invention is to provide the method that a kind of preparation has the heparin fragment that suppresses the smooth muscle cell proliferation activity.
Preparation provided by the present invention has the method for the heparin fragment that suppresses the smooth muscle cell proliferation activity, comprises the steps:
(1) with heparinase enzymolysis heparin, obtains the heparinase hydrolysis products;
(2) from described heparinase hydrolysis products, separate the heparin fragment that obtains having inhibition smooth muscle cell proliferation activity with the SPR sensing technology.
In the said process, describedly from described heparinase hydrolysis products, separate the method that obtains having the heparin fragment that suppresses the smooth muscle cell proliferation activity with the SPR sensing technology and comprise the steps:
1) Prostatropin of fixedly connected stimulation smooth muscle cell proliferation on chip obtains being connected with the chip of the Prostatropin that stimulates smooth muscle cell proliferation;
2) chip that described heparinase hydrolysis products and step 1) are obtained interacts, part in the described heparinase hydrolysis products and the Prostatropin specific combination on the described chip, the rest part not Prostatropin on described chip is combined, remove the part that the Prostatropin on described chip not is combined, obtain being connected with the chip with the heparinase hydrolysis products of Prostatropin specific combination;
3) with the heparinase hydrolysis products of described and Prostatropin specific combination from step 2) chip that obtains elutes, and namely obtains having the heparin fragment that suppresses the smooth muscle cell proliferation activity.
In the said process, the Prostatropin of described stimulation smooth muscle cell proliferation is human brain source Prostatropin.
In the said process, in the described step 3), in the described wash-out, used elutriant is that concentration is that sodium chloride aqueous solution or the concentration of 2M is the ammonium bicarbonate aqueous solution of 2M.
In the said process, described chip is connected with the CM5 chip of dextran for the surface.
In the said process, described method with heparinase enzymolysis heparin comprises the steps: in the described method, after described step (1), step (2) is preceding, comprise the step of described heparinase hydrolysis products being carried out following purifying: described heparinase hydrolysis products is carried out ultrafiltration with ultra-filtration membrane, get the heparinase hydrolysis products of molecular weight cut-off between 1000Da-8000Da.
In the said process, the method for described enzymolysis comprises the steps: described heparin is mixed with described heparinase, 25 ℃, the vibration condition under react; The rotating speed of described vibration is 100-200 rpm, is specially 150 rpms; The feed ratio of described heparin and described heparinase is the 10g heparin: the 1U heparinase.
In the said process, in the described step 1), described on chip the method for the Prostatropin of fixedly connected stimulation smooth muscle cell proliferation comprise the steps: to interact with solution and the described chip of the Prostatropin of described stimulation smooth muscle cell proliferation; The concentration of the Prostatropin solution of described stimulation smooth muscle cell proliferation is 10 mcg/ml;
Described step 2) in, the described interactional method of chip that described heparinase hydrolysis products and step 1) are obtained is that the solution of described heparinase hydrolysis products and chip that step 1) obtains are interacted; The concentration of heparinase hydrolysis products is 1mg/ml in the described heparinase hydrolysis products solution.
In the said process, in the described method, before described step 1), comprise the step with the dextran activation of described chip surface.
In the said process, described heparinase fermentation Sphingobacterium (Sphingobacterium sp.HC-6155) CGMCC NO.0660 obtains.
In the said process, described heparinase is to prepare according to the method that comprises the steps:
1) described fermentation Sphingobacterium (Sphingobacterium sp.HC-6155) CGMCC NO.0660 is inoculated in the fermention medium, cultivated centrifugal results thalline 40 hours for 30 ℃;
Described fermention medium is made up of NaCl, K
2HPO
4, MgSO
4, heparin, soyflour and water forms, the concentration of NaCl in substratum is 1 gram/L, K
2HPO
4Concentration in substratum is 2.5 gram/L, MgSO
4Concentration in substratum is 0.5 gram/L, and the concentration of heparin in substratum is 2 gram/L, and the concentration of soyflour in substratum is 2 gram/L.
2) with the homeo-osmosis liquid described thalline that suspends, centrifugal 20 minutes of 12,000r/min removes supernatant liquor; With the low salts solution described thalline that suspends, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again, and note is made supernatant liquor I; With the high level salt solution described thalline that suspends again, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again at last, and note is made supernatant liquor II; Merge described supernatant liquor I and described supernatant liquor II, obtain the heparinase crude enzyme liquid;
Low salts solution is with 2mmol MgSO
4Be dissolved in and obtain in 1L 20mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution;
High level salt solution is 300mmol NaCl to be dissolved in 10mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 1L obtain;
Homeo-osmosis liquid is the aqueous solution of 20% (quality percentage composition) sucrose.
3) described heparinase crude enzyme liquid is used SP Sepharose successively
TMXL strong cation exchange gel and SOURCE
TM30S strong cation exchange gel carries out purifying;
Use SP Sepharose
TMXL strong cation exchange gel carries out in the method for purifying, comprise following elution step: be that the phosphate buffered saline buffer of 0.12M is as initial liquid with NaCl concentration, be that the phosphate buffered saline buffer of 0.18M is as stop buffer with NaCl concentration, carry out linear gradient elution, elution time is 120 minutes, NaCl concentration gradient rate of change is constant in the described elution process, collects the 2ml elutriant at every turn, and collection NaCl concentration is the solution that the phosphate buffered saline buffer of 0.16M elutes;
Use SOURCE
TM30S strong cation exchange gel carries out in the method for purifying, comprise following elution step: the phosphate buffered saline buffer that with NaCl concentration is 0.05M is that the phosphate buffered saline buffer of 0.15M is as stop buffer as initial liquid, with NaCl concentration, carry out linear gradient elution, elution time is 120 minutes, NaCl concentration gradient rate of change is constant in the described elution process, each 2ml elutriant of collecting, collection NaCl concentration are the solution that the phosphate buffered saline buffer of 0.1M elutes;
The heparin fragment that suppresses the smooth muscle cell proliferation activity that has that is obtained by above-mentioned arbitrary described method also belongs to protection scope of the present invention.
The inventive method is based on the principle of affinity interaction between surface plasma resonance spectroscopic technique detection molecules, screening function specificity oligose fragment from the oligosaccharide mixture of high physiologically active and low side effect.This method has in real time, and non-marked and sensitive characteristics can filter out the oligosaccharides with physiological function really.The heparin fragment that the inventive method prepares has the function that suppresses smooth muscle cell proliferation, and when concentration was low, its inhibiting rate to smooth muscle cell all was higher than heparin; And with the heparin ratio, the heparin fragment that the present invention obtains has very low anticoagulating active, has only 1.9% of original heparin.Therefore, the inventive method and the heparin fragment for preparing can be used for clinical, have overcome the defective that heparin in use can cause tangible bleeding, make the practical application of the anti-smooth muscle cell proliferation performance of heparin become possibility.Therefore, the inventive method and product have broad application prospects.
Description of drawings
Fig. 1 is heparinase hydrolysis products collection of illustrative plates.
Fig. 2 is the anti-fetal umbilical smooth muscle cell proliferation activation analysis of heparinase hydrolysis products.
Fig. 3 is the anti-tumor activity analysis of heparinase hydrolysis products.
Fig. 4 is the saturated lotus root connection of Prostatropin.
Fig. 5 is heparinase hydrolysis products and chip surface factor interaction.
Fig. 6 is chip triple channel heparin-binding enzymolysis product process synoptic diagram simultaneously.
Fig. 7 is the efficient liquid phase chromatographic analysis of the heparin fragment of recovery.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Embodiment 1, heparin fragment and preparation thereof with inhibition smooth muscle cell proliferation activity
One, preparation has the heparin fragment that suppresses the smooth muscle cell proliferation activity
(1) heparinase enzymolysis heparin
Molecular weight cut-off is that 8,000 and 1,000 ultra-filtration membrane is all available from U.S. Pall company.
Heparin is available from Sigma company, and catalog number is H3393; This heparin product does not contain the peptide section, all is polysaccharide.
1, used heparinase is prepared as follows in this experiment:
Sphingobacterium (Sphingobacterium sp.HC-6155) CGMCC No.0660 (number of patent application: 02148066.4).
Bacterium fermentation: after the heparinase generation bacterial strain Sphingobacterium that the inclined-plane is preserved is activated, be inoculated in the liquid fermentation medium, cultivate after 40 hours the centrifugal results thalline of 5500r/min for 30 ℃.
Liquid fermentation medium is formed: by NaCl, K
2HPO
4, MgSO
4, heparin, soyflour and water forms, the concentration of NaCl in substratum is 1 gram/L, K
2HPO
4Concentration in substratum is 2.5 gram/L, MgSO
4Concentration in substratum is 0.5 gram/L, and the concentration of heparin in substratum is 2 gram/L, and the concentration of soyflour in substratum is 2 gram/L.
NaCl is available from modern east, Beijing fine chemicals company limited, K
2HPO
4Available from Chemical Reagent Co., Ltd., Sinopharm Group, MgSO
4Available from the Shantou Xilong Chemical Factory, Guangdong, heparin is available from Sigma company, and soyflour is available from Beijing bispin microbiological culture media products factory.
Low salts solution: with 2mmol MgSO
4Be dissolved in and obtain pH7.4 in 1L20mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution;
High level salt solution: 300mmol NaCl is dissolved in obtains pH7.4 in 10mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 1L.
With the method for osmotic shock, namely use homeo-osmosis liquid (20% aqueous sucrose solution), low salts solution and high level salt solution to handle cell successively, to discharge the heparinase in the cell pericentral siphon.Specific practice is as follows: at first use homeo-osmosis liquid suspension cell, centrifugal 20 minutes of 12,000r/min removes supernatant liquor; With low salts solution suspension cell, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again; With high level salt solution suspension cell again, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again at last.
The supernatant liquor that supernatant liquor and the high salt of above-mentioned less salt processing acquisition are handled acquisition merges as the heparinase crude enzyme liquid, uses SP Sepharose after the dialysis successively
TMXL strong cation exchange gel and SOURCE
TM30S strong cation exchange gel carries out purifying.Before the upper prop all the phosphate buffered saline buffer with 20mM pH7.4 carry out balance.In SP-Sepharose (Amersham Biosciences product), add the 2ml crude enzyme liquid, then in 0-120 minute time range, carrying out linear gradient elution with the phosphate buffered saline buffer that contains 0.12-0.18M NaCl (is that the phosphate buffered saline buffer of 0.12M is as initial liquid with NaCl concentration namely, be that the phosphate buffered saline buffer of 0.18M is as stop buffer with NaCl concentration, carry out linear gradient elution), NaCl concentration gradient rate of change is constant, each 2ml elutriant of collecting, be collected near the enzyme activity peak of 0.16M NaCl gradient, the liquid that elutes is dialysed and concentrated, as the initial sample of Source-30S (Amersham Biosciences product) chromatography; Among the Source-30S (Amersham Biosciences product), add the 2ml initial sample, then in 0-120 minute time range, carrying out linear gradient elution with the phosphate buffered saline buffer that contains 0.05-0.15M NaCl (is that the phosphate buffered saline buffer of 0.05M is as initial liquid with NaCl concentration namely, be that the phosphate buffered saline buffer of 0.15M is as stop buffer with NaCl concentration, carry out linear gradient elution), NaCl concentration gradient rate of change is constant, each 2ml elutriant of collecting, be collected near the enzyme activity peak of 0.1M NaCl gradient, desalination and concentrated obtains the heparinase powder.
Enzyme is lived and defined: per minute generates the required enzyme amount of the unsaturated uronic acid of 1 μ mol;
The heparinase powder dissolution in the phosphate buffered saline buffer of 20mM pH7.4, is obtained enzyme liquid.
The 2g heparin is dissolved in the phosphate buffered saline buffer of 100ml 20mM pH7.4, obtains the heparin substrate solution, the concentration of heparin in the heparin substrate solution is 20mg/ml.
The enzyme biopsy is surveyed: 0.1ml enzyme liquid and 0.1ml heparin substrate solution are mixed, and 36 ℃ of reactions with the hydrochloric acid end reaction of 1.8ml 50mM, were measured A232nm after 10 minutes.
Control tube: 0.1ml enzyme liquid and 0.1ml heparin substrate solution are mixed, add the hydrochloric acid of 1.8ml 50mM again, 36 ℃ of reactions were measured A232nm after 10 minutes.
As a result, the enzyme of heparinase powder is 0.5U/mg than living.
2, with above-mentioned heparinase enzymolysis heparin
The composition of heparin substrate solution: the 10g heparin is dissolved in the phosphate buffered saline buffer of 100ml 20mM pH7.4, obtains the heparin substrate solution.
Enzyme solution: (feed ratio of heparin and heparinase is 10g: the 1U heparinase), be positioned in the sealed vessel 25 ℃ of gentle vibrations of water-baths (rotating speed is 150 rpms) with 100ml heparin substrate solution and 20mg heparinase; The at interval variation of the 12 hours absorbance A232nms of sampling and measuring enzymolysis product under wavelength no longer changes (last light absorption value is 3.0) until the light absorption value of enzymolysis product.
Be the ultra-filtration membrane ultrafiltration of 8,000Da with the enzymolysis product molecular weight cut-off, get filtrate; Be the ultra-filtration membrane ultrafiltration of 1,000Da with the filtrate molecular weight cut-off, get and be trapped part; Obtain the component of molecular weight ranges between 1000Da-8000Da in the enzymolysis product, be the heparinase hydrolysis products, with the lyophilize of heparinase hydrolysis products, standby.
Heparin and enzymolysis product are carried out polyacrylamide gel electrophoresis respectively, and used gel strength is 20%, and after electrophoresis finished, the reddish black A dyeing with 0.08% was removed unnecessary dye liquor with distilled water flushing immediately up to there being clear band to occur, and the result as shown in Figure 1.Among Fig. 1, the 1st and the 4th swimming lane is the molecular distribution of heparin; The 3rd swimming lane is the molecular distribution of the heparinase hydrolysis products of enzyme process preparation.
Repetition, unanimity are as a result established in experiment 3 times.
(2) separate the heparin fragment with inhibition smooth muscle cell proliferation activity with the SPR sensing technology
1, fixedly connected Prostatropin on chip
Human brain source Prostatropin (heparin binding growth factor-2, basicbrain-derived growth factor, 17KDa)) available from Invitrogen company, catalog number is PHG0024;
The SPR sensing chip adopts the CM5 chip.
(1) activation of CM5 chip: inject N-hydroxy thiosuccinimide (NHS) that 35 μ L concentration are respectively 0.05mol/L and 0.2mol/L and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) successively to chip surface, flow velocity is 5 μ L/min, make the dextran activation of CM5 chip surface, obtain the surface and be the CM5 chip of the dextran molecule of activation.3 passages of CM5 chip all are activated processing, and note is made passage 1, passage 2, passage 3 respectively, and passage 4 does not activate in contrast.
(2) saturated fixedly human brain source Prostatropin (bFGF)
The concentration of human brain source Prostatropin solution is 10 mcg/ml;
The flow velocity that instrument is set is 10 microlitre per minutes, feed HBS damping fluid (pH 7.4) (PharmaciaBiosensor AB company) (solution composition is as follows: 10mmol/L 4-2 (2-hydroxyl) piperazine-1-ethylsulfonic acid, 150mmol/L NaCl, 3.4mmol/L EDTA, 0.005% (V/V) tensio-active agent P20) to chip surface, clean and obtain the balance baseline; Inject 5 microlitre bFGF solution in the surface that scribbles the dextran chip; Sample introduction no longer rises up to response value repeatedly, sealing, and passage 4 is passage in contrast.So chemical lotus root connection takes place in Prostatropin and the activated dextran molecule of chip surface, obtains the chip of saturated coupling bFGF, and the response value of 1,2,3 passages reaches about 5000RU (Fig. 4) respectively.(this response value is the poor of starting point and terminal point).
2, heparinase hydrolysis products and the Prostatropin combination of human brain source
(1) binding ability of heparinase hydrolysis products and cell growth factor detects: setting constant flow rate is 50 μ l/min, and sample is mixed with the series concentration gradient respectively, and the heparinase hydrolysis products is 0,3.9,7.8,31.3,62.5,125,250nM.Behind each sample introduction, (NaCl is dissolved in 100mM sodium acetatebuffer (pH 4.0) regeneration to chip surface with 50 μ l 2M NaCl solution.
As a result, heparin fragment is that obtain with the combination factor with dissociate characteristic spectrum figure as shown in Figure 5 under the different concns condition.A figure is the Heparin Oligosaccharides (being the heparinase hydrolysis products) of enzyme process preparation; B figure is chemical method Heparin Oligosaccharides (being the heparin split product).Two kinds of samples are as shown in table 1 with factor binding ability constant separately.k
aFor in conjunction with velocity factor, k
dBe the velocity factor that dissociates, KD is dissociation constant.The KD value is more little, illustrates that the binding ability of the two is more strong.
Table 1, various interactional dynamic analysis
*
| Interact | Association rate constant (M -1s -1) | Dissociation rate constant (s -1) | Dissociation constant (M) |
| Prostatropin/heparin | 2.6×10 6±3.25×10 4 | 4.72×10 -3±9.98×10 -5 | 1.82×10 -9 |
| Prostatropin/heparinase hydrolysis products | 1.85×10 6±4.57×10 4 | 1.07×10 -3±8.66×10 -5 | 5.82×10 -10 |
| Prostatropin/heparin split product | 2.69×10 5±3.14×10 3 | 7.72×10 -3±1.48×10 -4 | 2.87×10 -8 |
The above results shows: the binding ability of heparinase hydrolysis products and Prostatropin is the strongest.
(2) the heparinase hydrolysis products method of being combined with human brain source Prostatropin is as follows:
The heparinase hydrolysis products is made the solution that concentration is 1mg/ml.
Heparinase hydrolysis products solution is flow through simultaneously the passage 1 that has been combined with Prostatropin, 2,3, assurance heparinase hydrolysis products is combined with cytokine as much as possible, sample introduction repeatedly, make response value reach maximum, (solution composition is as follows: 10mmol/L4-2 (2-hydroxyl) piperazine-1-ethylsulfonic acid to use HBS damping fluid (pH 7.4) (Pharmacia Biosensor AB company) then, 150mmol/L NaCl, 3.4mmol/LEDTA, 0.005% (V/V) tensio-active agent P20) flushing chip surface, the heparinase hydrolysis products fragment that the flush away bonding force is more weak stays the heparinase hydrolysis products fragment of high-affinity.
Simultaneously heparin-binding enzymolysis product fragment process synoptic diagram are as shown in Figure 6A for chip channel 1,2,3. and A is sample introduction and in conjunction with the stage among the figure A, and the B stage is the unconjugated heparin fragment of flush away, and the C stage is that the high-bond fragment reclaims and the chip regenerative process.A and B stage for amplifying among the figure B.The response value of chip channel 1,2,3 heparin-binding enzymolysis products is shown in Fig. 6 B, and the response value of chip channel 1,2,3 heparin-binding enzymolysis product fragments is respectively 128.0,108.7,97.1.
The result shows: heparin enzymatic fragment and Prostatropin are saturated can to reach about 100 units in conjunction with the back response value, and multi-channel operation has good collimation, can be used for reclaiming function fragment simultaneously.
3, reclaim the heparin fragment with inhibition smooth muscle cell proliferation activity
The 2M sodium chloride aqueous solution is wash-out fully, and the 2M ammonium bicarbonate aqueous solution is wash-out fully also, but bicarbonate of ammonia is than the easy recovery of sodium-chlor, so adopt ammonium bicarbonate aqueous solution as the wash-out salts solution.
The wash-out recycling step: with the 2M ammonium bicarbonate aqueous solution with the velocity flow of 5um/min through being combined with the chip surface of heparin fragment, the stream that stops elutriant adding, make bicarbonate of ammonia and heparin fragment displacement 20-30 second, start stream then and add, the bicarbonate of ammonia that will contain heparin fragment is eluted in the specific container.All programs are carried out in 1,2,3 simultaneously.
The reclaimer of instrument is set, the heparinase fragment that elutes is recovered in the container.The wash-out reclaimer operation carries out in the passage 1,2,3 of chip simultaneously.Obtain having the heparin fragment that suppresses the smooth muscle cell proliferation activity.
(3) reclaim product and carry out efficient liquid phase chromatographic analysis
Preparation method with liquid phase sample of the heparin fragment that suppresses the smooth muscle cell proliferation activity: will reclaim product freeze-drying repeatedly, to remove bicarbonate of ammonia, then solids being dissolved in pH is in the 3.0-3.5 distilled water, obtains sample solution.
The preparation method of the liquid phase sample of heparin: it is the liquid phase sample that obtains heparin in the 3.0-3.5 distilled water that the 1mg heparin is dissolved in 1mlpH.
The condition of high performance liquid chromatography:
Chromatographic column is the half preparative high performance liquid chromatography post (SAX-HPLC) (available from the peculiar limit of Dalian Yi Li company) on the Waters 600 type liquid chromatographs; Column length 250mm, aperture 4.5mm, filled media is reinforcing yin essence ion exchange resin, aperture 5um; 25 ℃ of column temperatures;
The automatic sampler sample introduction, sample size 20ul;
Gradient eluent is: moving phase is the aqueous solution (pH 3.0-3.5) of 0-2M NaCl; Flow velocity is 1ml/min; The gradient elution time is 1h;
Detector is UV-detector, detects wavelength 232nm;
Under as above chromatographic condition, the high performance liquid phase with the heparin fragment that suppresses the smooth muscle cell proliferation activity is characterised in that their time that washes out is between 15 minutes to 25 minutes.The liquid chromatography of heparin sample correspondence is shown in Fig. 7 A, has the liquid chromatography of the heparin fragment sample correspondence that suppresses the smooth muscle cell proliferation activity shown in Fig. 7 B, the spectrum peak scope of primary sample is between 16-30 minute, and the spectrum peak of recovery sample mainly dropped between 16-20 minute.Show: have only the fragment that has a high-affinity with Prostatropin to obtain recovery in the heparin enzymatic fragment, the fragment that bonding force is low directly flows through chip surface, and combination does not take place.
Two, the function that has the heparin fragment that suppresses the smooth muscle cell proliferation activity
1, anticoagulating active detects
Heparin, heparinase hydrolysis products are carried out anticoagulating active respectively to be detected.
Method: carry out with reference to " heparin biologic assay " method in second one of the state-promulgated pharmacopoeia version in 2000.The anticoagulant active of the Heparin Oligosaccharides that measure to reclaim with rabbit plasma, and and the anticoagulating active of original heparin compare.
Result: the anticoagulating active 183.0U/mg of heparin; Heparinase hydrolysis products anticoagulating active 3.5U/mg.
Conclusion: heparinase hydrolysis products anticoagulant active reduces greatly than heparin, has only 1.9% of original heparin.
2, suppress the active detection of smooth muscle cell proliferation
(San Diego, USA, catalog number are 8030 to the fetal cord arterial smooth muscle cell available from ScienCell Research Laboratories; The smooth muscle cell complete culture solution is available from ScienCell ResearchLaboratories (San Diego, USA), and catalog number is 09211; Do not contain the substratum of foetal calf serum (10%FBS) available from ScienCell Research Laboratories (San Diego, USA), catalog number is 09011;
Adopt 96 orifice plates to cultivate the anti-proliferation of smooth muscle activity of fetal cord arterial smooth muscle cell and test Heparin Oligosaccharides.Concrete operations are as follows:
Heparin group: get the good logarithmic phase fetal cord arterial smooth muscle cell of upgrowth situation, the centrifugal cell that obtains after the trysinization, and carry out cell counting.Cell is dissolved in the smooth muscle cell complete culture solution again, diluting cells concentration to 2.5 * 10
4Cells/ml.By inoculating cell in 96 orifice plates of crossing, it is 2.5 * 10 that 200 μ l concentration are inoculated in every hole to bag
4The cell suspension of cells/ml.96 orifice plates behind the inoculating cell leave standstill 24h in incubator, the assurance smooth muscle cell is adherent fully and with substratum suction in 96 orifice plates to be gone, add 200 μ l and do not contain the substratum of foetal calf serum (10%FBS), add heparin again, the final concentration of heparin in the substratum that does not contain foetal calf serum (10%FBS) is 0mg/ml, 0.05mg/ml, 0.1mg/l, 0.2mg/l, 0.5mg/l, 1.0mg/l, each concentration is established 5 repeating holes, places cell culture incubator to cultivate 96 orifice plates after adding heparin, and heparin action time is 48 hours or 72 hours, carry out the MTT experiment then, measure the 492nm absorbancy.
Heparinase hydrolysis products group: identical with heparin group, different is that heparin is replaced to the heparinase hydrolysis products, and the final concentration in the substratum that does not contain foetal calf serum (10%FBS) of heparinase hydrolysis products is 0mg/ml, 0.05mg/ml, 0.1mg/l, 0.2mg/l, 0.5mg/l, 1.0mg/l.
Control group: method is identical with heparin group, and different is not add heparin.
The calculation formula of inhibiting rate:
Inhibiting rate (%)=(B-A) 100%/A; A be control group at the light absorption value at OD492nm place, B is that each experimental group is at the light absorption value of OD492nm.
3 repetitions are established in experiment.3 repetitions, results averaged ± standard deviation are established in experiment.The result as shown in Figure 2.
The result shows: when adding consistency was 0.05mg/ml, heparin was 13.1 ± 5.7% to the inhibiting rate of smooth muscle cell, and the heparinase hydrolysis products is 17.6 ± 5.4% to the inhibiting rate of smooth muscle cell; When adding consistency was 0.2mg/ml, heparin was 18.1 ± 4.9% to the inhibiting rate of smooth muscle cell, and the heparinase hydrolysis products is 20.3 ± 6.6% to the inhibiting rate of smooth muscle cell; When adding consistency was 0.5mg/ml, heparin was 32.4 ± 5.1% to the inhibiting rate of smooth muscle cell, and the heparinase hydrolysis products is 25.2 ± 1.6% to the inhibiting rate of smooth muscle cell.
Through statistical analysis, show: heparinase hydrolysis products and heparin are under the same medicine condition, and the activity that suppresses smooth muscle cell proliferation does not have significant difference, illustrate that heparinase hydrolysis products that enzyme process prepares has kept the anti-proliferation of smooth muscle activity of original heparin preferably.
3, anti-tumor activity analysis
Experimental group: 1) at 37 ℃, 5%CO
2Under the condition of concentration, static cultivation Hela cell changed a not good liquor in average two days, passed once generation in four days.2) inoculating cell: be made into 10 with complete culture solution
5The cell suspension of/ml, with the volume of every hole 600ul with the logarithmic phase cell inoculation to 24 orifice plates.One group of contrast, nine experimental group, every group of three holes.3) culturing cell: 37 ℃, 5%CO
2Under the condition of concentration, cultivate 24h, make cell adapted new environment.4) dosing: distinguish dosing in each hole, the final concentration of each medicine in substratum is respectively 0mg/l, 100mg/l, 250mg/l, 500mg/l; Medicine is heparin, heparinase hydrolysis products.5) colour developing: cultivate after 48h days, abandon nutrient solution, wash once with PBS.Every hole adds the MTT solution 600ul of 0.5mg/ml.Continued to hatch 4 hours, and stop to cultivate, abandon supernatant liquor after suspension cell is centrifugal.Every hole adds 450ul methyl-sulphoxide (DMSO), and vibration is fully melted crystallisate.6) colorimetric: select the 492nm wavelength, measure each hole absorbance value at the enzyme linked immunological monitor, the record result.Be X-coordinate with the final concentration of medicine in substratum, inhibiting rate is that ordinate zou is drawn cell growth curve.
Control group: method is identical with experimental group, and different is not add heparin, heparinase hydrolysis products.
The calculation formula of inhibiting rate is:
Inhibiting rate (%)=(B-A) 100%/A; A be control group at the light absorption value at OD492nm place, B is that each experimental group is at the light absorption value of OD492nm.
3 repetitions, results averaged ± standard deviation are established in experiment.The result as shown in Figure 3.
The result shows: when adding consistency was 100mg/l, heparin was 28.2 ± 8.29% to Hela cell inhibiting rate, and the heparinase hydrolysis products is 0 ± 8.14% to Hela cell inhibiting rate; When adding consistency was 250mg/l, heparin was 34.4 ± 5.88% to Hela cell inhibiting rate, and the heparinase hydrolysis products is 13.5 ± 8.48% to Hela cell inhibiting rate; When adding consistency was 500mg/l, heparin was 38.4 ± 10.04% to Hela cell inhibiting rate, and the heparinase hydrolysis products is 15.2 ± 7.56% to Hela cell inhibiting rate;
Determination data is carried out statistical analysis, show, the heparinase hydrolysis products has lost most inhibition activity of tumor cells.
Claims (7)
1. one kind prepares the method with the heparin fragment that suppresses the smooth muscle cell proliferation activity, comprises the steps:
(1) with heparinase enzymolysis heparin, obtains the heparinase hydrolysis products;
(2) from described heparinase hydrolysis products, separate the heparin fragment that obtains having inhibition smooth muscle cell proliferation activity with the SPR sensing technology; Describedly from described heparinase hydrolysis products, separate the method that obtains having the heparin fragment that suppresses the smooth muscle cell proliferation activity with the SPR sensing technology and comprise the steps:
1) Prostatropin of fixedly connected stimulation smooth muscle cell proliferation on chip obtains being connected with the chip of the Prostatropin that stimulates smooth muscle cell proliferation;
2) chip that described heparinase hydrolysis products and step 1) are obtained interacts, part in the described heparinase hydrolysis products and the Prostatropin specific combination on the described chip, the rest part not Prostatropin on described chip is combined, remove the part that the Prostatropin on described chip not is combined, obtain being connected with the chip with the heparinase hydrolysis products of Prostatropin specific combination;
3) with the heparinase hydrolysis products of described and Prostatropin specific combination from step 2) chip that obtains elutes, and namely obtains having the heparin fragment that suppresses the smooth muscle cell proliferation activity;
Described heparinase fermentation Sphingobacterium (Sphingobacterium sp.) HC-6155CGMCC NO.0660 obtains; The method of described enzymolysis comprises the steps: described heparin is mixed with described heparinase, 25 ℃, the vibration condition under react, the rotating speed of described vibration is 100-200 rpm.
2. method according to claim 1, it is characterized in that: the Prostatropin of described stimulation smooth muscle cell proliferation is human brain source Prostatropin; In the described step 3), in the described wash-out, used elutriant is that concentration is that sodium chloride aqueous solution or the concentration of 2M is the ammonium bicarbonate aqueous solution of 2M.
3. method according to claim 1 and 2 is characterized in that: described chip is connected with the CM5 chip of dextran for the surface; In the described method, before described step 1), comprise the step with the dextran activation of described chip surface.
4. method according to claim 3, it is characterized in that: in the described method, after described step (1), step (2) is preceding, comprise the step of described heparinase hydrolysis products being carried out following purifying: described heparinase hydrolysis products is carried out ultrafiltration with ultra-filtration membrane, get the heparinase hydrolysis products of molecular weight between 1000Da-8000Da.
5. method according to claim 4 is characterized in that:
The feed ratio of described heparin and described heparinase is the 10g heparin: the 1U heparinase.
6. method according to claim 5, it is characterized in that: in the described step 1), described on chip the method for the Prostatropin of fixedly connected stimulation smooth muscle cell proliferation comprise the steps: to interact with solution and the described chip of the Prostatropin of described stimulation smooth muscle cell proliferation; The concentration of the Prostatropin of the Prostatropin solution moderate stimulation smooth muscle cell proliferation of described stimulation smooth muscle cell proliferation is 10 mcg/ml;
Described step 2) in, the described interactional method of chip that described heparinase hydrolysis products and step 1) are obtained is that the solution of described heparinase hydrolysis products and chip that step 1) obtains are interacted; The concentration of heparinase hydrolysis products is 1mg/mL in the solution of described heparinase hydrolysis products.
7. method according to claim 6, it is characterized in that: described heparinase is to prepare according to the method that comprises the steps:
1) described fermentation Sphingobacterium (Sphingobacterium sp.) HC-6155CGMCC NO.0660 is inoculated in the fermention medium, cultivated centrifugal results thalline 40 hours for 30 ℃;
Described fermention medium is by NaCl, K
2HPO
4, MgSO
4, heparin, soyflour and water forms, the concentration of NaCl in fermention medium is 1 gram/L, K
2HPO
4Concentration in fermention medium is 2.5 gram/L, MgSO
4Concentration in fermention medium is 0.5 gram/L, and the concentration of heparin in fermention medium is 2 gram/L, and the concentration of soyflour in fermention medium is 2 gram/L;
2) with the homeo-osmosis liquid described thalline that suspends, centrifugal 20 minutes of 12,000r/min removes supernatant liquor; With the low salts solution described thalline that suspends, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again, and note is made supernatant liquor I; With the high level salt solution described thalline that suspends again, centrifugal 20 minutes of 12,000r/min collects supernatant liquor again at last, and note is made supernatant liquor II; Merge described supernatant liquor I and described supernatant liquor II, as the heparinase crude enzyme liquid;
Low salts solution is with 2mmol MgSO
4Be dissolved in and obtain in 1L20mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution; The pH value of described low salts solution is 7.4;
High level salt solution is 300mmol NaCl to be dissolved in 10mM Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution of 1L obtain; The pH value of described high level salt solution is 7.4;
Homeo-osmosis liquid is the aqueous solution of 20% (quality percentage composition) sucrose;
3) described heparinase crude enzyme liquid is used SP Sepharose successively
TMXL strong cation exchange gel and SOURCE
TM30S strong cation exchange gel carries out purifying;
Use SP Sepharose
TMXL strong cation exchange gel carries out in the method for purifying, comprise following elution step: be that the phosphate buffered saline buffer of 0.12M is as initial liquid with NaCl concentration, be that the phosphate buffered saline buffer of 0.18M is as stop buffer with NaCl concentration, carry out linear gradient elution, elution time is 120 minutes, NaCl concentration gradient rate of change is constant in the described elution process, collects the 2mL elutriant at every turn, and collection NaCl concentration is the solution that the phosphate buffered saline buffer of 0.16M elutes;
Use SOURCE
TM30S strong cation exchange gel carries out in the method for purifying, comprise following elution step: the phosphate buffered saline buffer that with NaCl concentration is 0.05M is that the phosphate buffered saline buffer of 0.15M is as stop buffer as initial liquid, with NaCl concentration, carry out linear gradient elution, elution time is 120 minutes, NaCl concentration gradient rate of change is constant in the described elution process, each 2mL elutriant of collecting, collection NaCl concentration are the solution that the phosphate buffered saline buffer of 0.1M elutes.
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| CN2010191140713A CN102146425B (en) | 2010-02-08 | 2010-02-08 | Heparin fragment with activity of inhibiting smooth muscle cell proliferation and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2010191140713A CN102146425B (en) | 2010-02-08 | 2010-02-08 | Heparin fragment with activity of inhibiting smooth muscle cell proliferation and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102146425A CN102146425A (en) | 2011-08-10 |
| CN102146425B true CN102146425B (en) | 2013-07-10 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429913A (en) * | 2001-12-30 | 2003-07-16 | 中国科学院微生物研究所 | Method of producing heparin oligosaccharide using heparinase |
| CN101568836A (en) * | 2006-10-24 | 2009-10-28 | Rna控股有限公司 | Immobilisation and application of antigenic carbohydrates to detect infective micro-organisms |
| CN102040671A (en) * | 2009-10-13 | 2011-05-04 | 北京贯虹科技有限公司 | Process for preparing and purifying ultra low molecular weight heparin |
-
2010
- 2010-02-08 CN CN2010191140713A patent/CN102146425B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429913A (en) * | 2001-12-30 | 2003-07-16 | 中国科学院微生物研究所 | Method of producing heparin oligosaccharide using heparinase |
| CN101568836A (en) * | 2006-10-24 | 2009-10-28 | Rna控股有限公司 | Immobilisation and application of antigenic carbohydrates to detect infective micro-organisms |
| CN102040671A (en) * | 2009-10-13 | 2011-05-04 | 北京贯虹科技有限公司 | Process for preparing and purifying ultra low molecular weight heparin |
Non-Patent Citations (2)
| Title |
|---|
| 丛祥凤等.肝素小分子片段对平滑肌细胞生长及其抗凝作用的对比研究.《中华心血管病杂志》.2001,第29卷(第04期),250. |
| 肝素小分子片段对平滑肌细胞生长及其抗凝作用的对比研究;丛祥凤等;《中华心血管病杂志》;20010428;第29卷(第04期);250 * |
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| CN102146425A (en) | 2011-08-10 |
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