CN101824100A

CN101824100A - Heparin-12-sugar, preparation method thereof and application thereof in resisting vascular smooth muscle cell proliferation

Info

Publication number: CN101824100A
Application number: CN 201010139398
Authority: CN
Inventors: 何书英; 孙程琛; 牛佳; 高婷; 李巍; 刘亚梅; 吴梧桐
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2010-04-02
Filing date: 2010-04-02
Publication date: 2010-09-08
Anticipated expiration: 2030-04-02
Also published as: CN101824100B

Abstract

The invention relates to the field of biological medicaments, in particular to a heparin-derived oligosaccharide-12-polymer, a preparation method thereof, and application thereof in resisting vascular smooth muscle cell proliferation. The heparin-derived oligosaccharide-12-polymer has a structural formula (I) as follows.

Description

The purposes of a kind of heparin-12-sugar, its method for making and resisting vascular smooth muscle cell proliferation thereof

Technical field

The present invention relates to biomedicine field, be specifically related to a kind of Heparin Oligosaccharides ten dimers, it has the effect of resisting vascular smooth muscle cell proliferation.

Background technology

The mucopolysaccharide class mixture with different chain length that heparin is made up of uronic acid and hexosamine, its molecular weight is 3000-30000.It not only has multiple biological functions such as anti-freezing, antithrombotic, anti-inflammatory, antianaphylaxis, antiviral and reducing blood-fat, also is a kind of active drug of preventing and treating dvt, pulmonary infarction or other thrombus simultaneously.But heparin often with side effects such as hemorrhage, thrombopenia, osteoporosises, sometimes even can cause that lethality intracranials hemorrhage, it is reported that the hemorrhage rate that is caused by heparin is up to 35% in clinical application.

Low molecular heparin molecular-weight average 5kD, it is to be got by the unfractionated heparin depolymerization.Compare with unfractionated heparin, Low molecular heparin has strong, the anti-FIIa effect of anti-FXa effect is weak, bioavailability is high, plasma half-life is long, lower bleeding tendency and less advantages such as thrombocytopenia, can be applied to prevent and treat diseases such as dvt, pulmonary infarction, disseminated inravascular coagulation.

But Low molecular heparin is a mixture, Heparin Oligosaccharides by different polymerization degree is formed, find at present it specifically is that the Heparin Oligosaccharides of which polymerization degree can the vasoactive smooth muscle cell as yet, so this research is carried out purifying to Heparin Oligosaccharides, and it is suppressed the vascular smooth muscle cell proliferation activity study, this research meets China's medicine development of modernization direction, has great social significance and potential economic benefit in fundamental research field and applied research field.

Summary of the invention

The invention discloses a kind of Heparin Oligosaccharides ten dimers, its structural formula is as follows:

Heparin Oligosaccharides ten dimers of the present invention can prepare with following method:

Prepare the small molecules heparin earlier, can utilize the method for bibliographical information, also can prepare: use reaction buffer (25mM NaAc-10mM CaAc2, pH7.0) dissolving heparin sodium with following method, 30 ℃ of enzyme process prepare Low molecular heparin, and ultrafiltration obtains the small molecules heparin of molecular-weight average at 4kD.

Get the lyophilized powder of above-mentioned molecular weight less than the Low molecular heparin of 10kD, the dissolving of use moving phase, 0.45 μ m millipore filtration adopts Bio-Gel P6 gel chromatographic columns to separate, parting liquid carries out electrophoresis detection again, the molecular weight that electrophoresis obtains Heparin Oligosaccharides divides five ranks from low to high, the Heparin Oligosaccharides merging of second level is concentrated, after Sephadex G15 desalination, adopt Bio-Gel P4 gel chromatographic columns to separate, and use the gradient electrophoresis checking, promptly.

The preferred 0.2M sodium chloride solution of the isolating moving phase of Bio-Gel P6 gel chromatographic columns wherein.

The preferred 0.4M sodium chloride solution of the isolating moving phase of Bio-Gel P4 gel chromatographic columns wherein.

The all preferred 1mL/4min of flow velocity.

Wherein the concentration of the separation gel of gradient electrophoresis is 15-25% (w/v), and last sample concentration is 2mg/ml.

Heparin Oligosaccharides ten dimers of the present invention can add water for injection and be prepared into injection liquid or lyophilized injection, or add the acceptable accessories composition be prepared into formulation pharmaceutically commonly used be used for clinical, as Heparin Oligosaccharides ten dimer injection liquids or Heparin Oligosaccharides ten dimer lyophilized injections etc.

External activity experimental results show that the effect excellence of Heparin Oligosaccharides ten dimer resisting vascular smooth muscle cell proliferations of the present invention.Inducing in the model of proliferation of smooth muscle with the DMEM (Dulbecco Modified Eagle Medium) that contains 20% calf serum, after cultivating 24 hours with Heparin Oligosaccharides ten dimers of 0.01,01,1 μ mol/L, it is obvious to containing 20% serum DMEM inductive hyperplasia restraining effect.And other Heparin Oligosaccharides components do not have definite conclusion to the influence that contains serum DMEM inductive smooth muscle cell proliferation.

Be part pharmacology test and result below:

One, former generation arterial smooth muscle cultivation

Get new calves neck aorta,, after the D-Hanks balanced salt solution cleans, divest inner membrance, expose the middle level unstriated muscle with curved tweezer with containing the antibiotic Hanks liquid of high density.Unstriated muscle is cut into small pieces, is inoculated in the culturing bottle, add the DMEM that 6～7mL contains 20% new-born calf serum, will put into 37 ℃ at the bottom of the culturing bottle bottle up, 5%CO ₂Incubator is cultivated 4h, treats that tissue block firmly attaches to a bottle wall, and the upset culturing bottle makes tissue block be immersed in nutrient solution fully, continues to cultivate, and changes one time nutrient solution in per 3 days.Most of regional cell can go down to posterity near merging tangible " peak-paddy " the staggered form of formation at the bottom of bottle.3～5 generation VSMC are adopted in experiment.

Two, external anti-smooth muscle cell proliferation test

Sample according to three groups of each minutes of concentration (0.01,0.1,1 μ mol/L), is established 12 multiple holes, adopted tetrazole indigo plant (MTT) method to detect for every group.Cell is divided into blank group (DMEM that does not contain serum), control group (DMEM that contains 20% new-born calf serum), dosing group (DMEM that contains 20% new-born calf serum), establish 12 multiple holes for every group, blank group, control group add the PBS of respective amount, the dosing group adds the heparin-12-sugar of 0.01,0.1,1 μ mol/L respectively, continues to cultivate 24 hours.Every hole adds the MTT solution 20 μ L of 5mg/ml, cultivates 4h for 37 ℃.The sucking-off nutrient solution, every hole adds DMSO 150 μ L, behind the vibration mixing in 490nm place mensuration absorbancy.All data statistics differences adopt Student ' s t-test to analyze.P＜0.01 is for there being utmost point significant difference.Detected result sees Table 1.

Table 1 heparin-12-sugar to the influence of smooth muscle cell proliferation (

N=12)

^##Compare t＜0.01 with the blank group, ^*Compare t＜0.01 with control group

Description of drawings

Fig. 1 is the ultraviolet absorption curve of 232nm place elutriant.

Fig. 2 is the electrophoresis result of the Low molecular heparin for preparing of the present invention through Bio-Gel P6 initial gross separation.

Fig. 3 is the heparin-12-sugar polyacrylamide gel electrophoresis figure that the present invention prepares.

Embodiment

Embodiment 1

1) enzyme process prepares low molecular weight heparin

Taking heparin sodium 50mg, use reaction buffer (25mM NaAc-10mM CaAc2, pH7.0) dissolve, 30 ℃ of water bath with thermostatic control 10min add the Heparinase I 26125mU (heparin sodium: Heparinase I=1: 522.5) carry out the constant temperature enzyme digestion reaction available from Sigma company.The Heparinase I of respectively adding equivalent in 8,16,24 hours in reaction is once added three times altogether, with the enzyme loss in preventing to react.Control was reflected at about 32 hours, boiled in 100 ℃ boiling water 4 minutes, and deactivation Heparinase I termination reaction obtains the reaction solution of Heparin Oligosaccharides.Get above-mentioned reaction solution, centrifugal (5000r/min * 30min) carries out ultrafiltration in refrigerated centrifuge with the ultra-filtration centrifuge tube of 10kD MWCO, the sucking-off of centrifuge tube outer tube liquid, interior pipe adds distilled water, and repeated centrifugation twice merges the outer component less than 10kD of pipe, use the filter membrane of 0.45 μ m to filter, 50 ℃ of vacuum are revolved steaming, and lyophilize obtains the active stronger Low molecular heparin relatively of anti-smooth muscle cell proliferation.

2) the initial gross separation purifying of Heparin Oligosaccharides

Get above-mentioned lyophilized powder, with the moving phase dissolving, (moving phase is 0.2mol/L NaCl for Bio-Gel P6,1.0cm * 100cm) separate, and flow velocity is 1mL/4min, and every pipe is collected 2mL to adopt gel chromatographic columns.Through 232nm place ultraviolet detection, ultraviolet absorption curve figure sees Fig. 1.

3) polyacrylamide gel electrophoresis

Get the collection liquid 10 μ L (go up sample concentration and be about 2mg/ml) at each absorption peak place of above-mentioned ultraviolet absorption curve,, carry out electrophoresis as indicator with tetrabromophenol sulfonphthalein with isopyknic 40% sucrose solution mixing.The concentration of running gel adopts the gradient separations glue of 15%-25%, 5% concentrated glue.Behind the 150V electrophoresis 1 hour, regulating voltage is to 200V, when the tetrabromophenol sulfonphthalein migration distance is to stop electrophoresis two of separation gel/a period of time.Put the 30min that dyes in 0.25% (w/v) A Lixinlan staining fluid, take off to background colourless with 1% (v/v) acetic acid.Electrophoresis result shows that the molecular weight of Heparin Oligosaccharides divides five ranks from low to high, sees Fig. 2.

4) separation and purification of heparin-12-sugar

The Heparin Oligosaccharides merging of second level is concentrated, and after Sephadex G15 desalination, (moving phase is 0.4mol/LNaCl for Bio-Gel P4,1.0cm * 100cm) separate, and flow velocity is 1mL/4min, and every pipe is collected 1mL to adopt gel chromatographic columns.The SephadexG15 desalination, freeze-drying.Because second component contains two kinds of oligosaccharides, be respectively heparin-12-sugar and heparin ten tetroses, wherein the molecular weight of heparin ten tetroses is greater than 4000D, and the separating ranges of Bio-Gel P4, is directly eluted so heparin ten tetroses can not enter the space of gel media substantially at 1000-4000, and the heparin-12-sugar molecular weight is less, can enter the space of Bio-Gel P4, according to this principle, thereby realization is to the separation of two kinds of oligosaccharides.Determine heparin-12-sugar by the 15-25% polyacrylamide gel electrophoresis.See Fig. 3.

Claims

1. Heparin Oligosaccharides ten dimers of a structural formula (I):

2. the Heparin Oligosaccharides ten dimeric preparation methods of a claim 1, comprise: get Low molecular heparin and adopt Bio-Gel P6 gel chromatographic columns to separate, parting liquid carries out electrophoretic separation again, the molecular weight that electrophoresis obtains Heparin Oligosaccharides divides five ranks, second component in the Heparin Oligosaccharides is concentrated, after Sephadex G15 desalination, adopt Bio-Gel P4 gel chromatographic columns to separate, promptly.

3. the preparation method of claim 2, wherein: the isolating moving phase of Bio-Gel P6 gel chromatographic columns is the 0.2M sodium chloride solution.

4. the preparation method of claim 2, wherein the isolating moving phase of Bio-Gel P4 gel chromatographic columns is the 0.4M sodium chloride solution.

5. the preparation method of claim 2, wherein the concentration of electrophoretic separation gel is 15-25%w/v, last sample concentration is 2mg/ml.

6. pharmaceutical composition wherein contains Heparin Oligosaccharides ten dimers and the pharmaceutically acceptable carrier of claim 1.

7. Heparin Oligosaccharides ten dimers of claim 1 are used to prepare the purposes of the medicine for the treatment of the smooth muscle cell proliferation relative disease.

8. the purposes of claim 7, wherein the smooth muscle cell proliferation relative disease is an atherosclerosis.