CN103408677A - Periodate-oxidized heparan sulfate for inhibiting cell proliferation - Google Patents
Periodate-oxidized heparan sulfate for inhibiting cell proliferation Download PDFInfo
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- CN103408677A CN103408677A CN2013103826358A CN201310382635A CN103408677A CN 103408677 A CN103408677 A CN 103408677A CN 2013103826358 A CN2013103826358 A CN 2013103826358A CN 201310382635 A CN201310382635 A CN 201310382635A CN 103408677 A CN103408677 A CN 103408677A
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- derivative
- heparan sulfate
- suleparoid
- periodate
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Abstract
The invention discloses a heparan sulfate derivative prepared from heparan sulfate as a raw material by utilizing a periodate oxidation method. The method comprises the following steps of: dissolving the heparan sulfate in water; adding a sodium periodate aqueous solution with a certain concentration; regulating a pH value to 5.2; fully mixing the mixture; standing the mixture in a dark place for reacting for 24 hours at 4 DEG C; adding ethylene glycol to stop the reaction; dialyzing the mixture for 72 hours at 4 DEG C with distilled water; freeze-drying and recycling an obtained sample; dissolving the oxidized heparan sulfate in a small amount of water; adding a certain amount of sodium borohydride solid under a stirring condition; regulating the pH value to 3.0 with hydrochloric acid; neutralizing the mixture with sodium hydroxide after reacting for 3 hours; dialyzing the mixture for 72 hours at 4 DEG C with the distilled water; and freeze-drying and recycling the sample, thereby obtaining the periodate-oxidized heparan sulfate. The heparan sulfate derivative has the activity of inhibiting the proliferation activity of cancer cells and umbilical vein endothelial cells.
Description
Technical field
The present invention relates to a kind of Suleparoid (Heparan Sulfate, HS) of periodate oxidation, the application of its preparation method and inhibition cell proliferation aspect.
Background technology
Suleparoid (Heparan Sulfate, HS) claim again glucosaminoglycan (Glycosaminoglycan, GAG), to have electronegative macromole linear polymer, by glucuronic acid (GlcA) and glucosamine (Glucosamine) disaccharide unit, formed by the glycosidic link repeated polymerization, its structure such as Fig. 1 [Khan S, Rodriguez E, Patel R, Gor J, Mulloy B, Perkins S. The Solution Structure of Heparan Sulfate Differs from that of Heparin. J Biol Chem. 2011, 286 (28): 24842 – 24854].HS extensively is present in Mammals and some plant materialss, and the complicacy of its structure and diversity are the bases of various vital movements.
Studies confirm that in a large number, HS has stronger neoplasm growth activity, and its mechanism of action mainly comprises inhibition tumor cell propagation and tumor-blood-vessel growth two aspects.Special site on the HS backbone is the key of its energy inhibition tumor cell propagation.The more important thing is, HS can be combined with multiple somatomedin.Many and the endothelial cell proliferation of these somatomedins, induce the capillary vessel blastogenesis, increase capillary permeability, promote cell migration and inhibited apoptosis etc. movable closely related, HS is combination with it, thereby block the signal path of its expression and the ability of modulating vascular growth, and then suppress tumor-blood-vessel growth.
In view of the activity performance of HS at anti-tumor aspect, also increasing for the research of HS analog in recent years.Periodate oxidation is a kind of Polysaccharide Modification method, document [Lapierre F is arranged, Holme K, Lam L, Tressler RJ, Storm N, Wee J, Stack RJ, Castellot J, Tyrrell DJ. Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties. Glycobiology. 1996, 6 (3): 355-366.] the report heparin is after periodate oxidation is modified, improved its anti-tumor activity.But up to now, about periodate oxidation, do not modify the relevant report of HS, and the present invention provides a kind of HS of periodate oxidation first, it has the activity of inhibition tumor cell propagation.
Summary of the invention
The invention provides a kind of HS of periodate oxidation, its molecular weight M
w=14063 Da, molecular weight distribution is 1277～45951 Da.
The present invention also provides the preparation method of a kind of HS of periodate oxidation:
Get HS 0.1～2.0 g, swell in 10～40 mL water, add 10～40 mL 0.05～0.5 mol/L NaIO
4, adjust pH is 5.2, fully mixes, and is placed in dark place in 4 ℃ of reaction 24 h, add the ethylene glycol termination reaction,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, HS after oxidation is dissolved in a small amount of water, under agitation condition, adds approximately 100 mg solid NaBH
4, with the HCl adjust pH, be 3.0, reaction 3 h, then, with the NaOH neutralization,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, make the HS of periodate oxidation.
Described reaction conditions is preferably 0.2～1.0 g HS and swells in 20～30 mL water, adds 20～30 mL 0.1～0.3 mol/L NaIO
4, regulating the pH value is 5.2, fully mixes, and is placed in dark place in 4 ℃ of reaction 24 h, more preferably 0.5 g HS swells in 20 mL water, adds 20 mL 0.2 mol/L NaIO
4.
The present invention also provides the purposes of HS in inhibition tumor cell and endothelial cell proliferation of above-mentioned periodate oxidation.Inhibition tumor cell proliferation activity of the present invention can be used to comprising treatment and prophylaxis of tumours and transfer thereof and recurrence.
The accompanying drawing explanation
Fig. 1 A and B be respectively HS variable disaccharide sequence (R=Ac, H or
R
1=Ac, H or
, R and R
1Can be the same or different).
Fig. 2 is the high-efficient liquid phase chromatogram that the high performance liquid phase gel exclusion chromatography is measured the HS molecular weight of periodate oxidation.
Embodiment
Below the invention will be further described by specific embodiment, but protection content of the present invention is not limited to following examples.
Embodiment 1
Get HS 0.5 g, swell in 20 mL water, add 20 mL 0.2 mol/L NaIO
4, adjust pH is 5.2, fully mixes, and is placed in dark place in 4 ℃ of reaction 24 h, add the ethylene glycol termination reaction,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, HS after oxidation is dissolved in a small amount of water, under agitation condition, adds approximately 100 mg solid NaBH
4, with the HCl adjust pH, be 3.0, reaction 3 h, then, with the NaOH neutralization,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, make the HS of periodate oxidation.
Embodiment 2
The molecular weight determination of the HS of periodate oxidation adopts the high performance liquid phase gel exclusion chromatography.Moving phase is 0.1 mol/L ammonium acetate and 0.02% sodiumazide mixing solutions, the molecular weight reference substance is heparin sodium (NIBSC code 07/324), chromatography in series post TSK Guard column (7.8mm * 30cm), TSK SW xl4000 (7.8mm * 30cm) and TSK SW xl3000 (7.8mm * 30cm), 30 ℃ of column temperatures, elution flow rate is 0.6 mL/min, differential refraction detector, temperature are 30 ℃.The typical curve of GPC Software on Drawing heparin sodium reference substance molecular weight and retention time is: LogM
w=1.09e
+ 001-5.06e
-001T+1.41e
-002T
2-1.55e
-004T
3(R
2=9999).The HS molecular weight M of GPC computed in software periodate oxidation
w=14063 Da, molecular weight distribution is 1277～45951 Da, and the component that molecular weight is greater than 20000 Da accounts for 16.35%, and the component that molecular weight is less than 8000 Da accounts for 19.00%.
Embodiment 3
Get 96 porocyte culture plates, every hole adds respectively approximately 5 * 10
3Individual human liver cancer cell (HepG2), in 5% CO
2In incubator, cultivate 12 h for 37 ℃, add respectively the DMEM substratum of the HS that contains 1 mg/mL and 0.1 mg/mL periodate oxidation; The DMEM substratum of separately take is blank, at CO
2In incubator, cultivate 48h, every hole adds 10 μ L MTT, after hatching 4 h, sucks substratum, adds 150 μ L DMSO, and after fully shaking up, 490 nm detect on the enzyme immunoassay instrument, and the sample light absorption value is A
1, blank absorbency is A
2, inhibiting rate (%)=
.The HS that in concentration is 1 mg/mL and 0.1 mg/mL periodate oxidation is 57.88% and 30.65% to the HepG2 inhibiting rate.
Embodiment 4
Get 96 porocyte culture plates, every hole adds respectively approximately 5 * 10
3Individual human cervical carcinoma cell lines (HeLa), in 5% CO
2In incubator, cultivate 12 h for 37 ℃, add respectively the DMEM substratum of the HS that contains 1 mg/mL and 0.1 mg/mL periodate oxidation; The DMEM substratum of separately take is blank, at CO
2In incubator, cultivate 48 h, every hole adds 10 μ L MTT, after hatching 4 h, sucks substratum, adds 150 μ L DMSO, and after fully shaking up, 490 nm detect on the enzyme immunoassay instrument, and the sample light absorption value is A
1, blank absorbency is A
2, inhibiting rate (%)=
.The HS that in concentration is 1 mg/mL and 0.1 mg/mL periodate oxidation is 51.73% and 42.21% to the HeLa inhibiting rate.
Embodiment 5
Get 96 porocyte culture plates, every hole adds respectively approximately 5 * 10
3Individual Human umbilical vein endothelial cells (HUVEC), in 5% CO
2In incubator, cultivate 12 h for 37 ℃, add respectively the ECM substratum of the HS that contains 1 mg/mL and 0.25 mg/mL periodate oxidation; The ECM substratum of separately take is blank, at CO
2In incubator, cultivate 48 h, every hole adds 10 μ L MTT, after hatching 4 h, sucks substratum, adds 150 μ L DMSO, and after fully shaking up, 490 nm detect on the enzyme immunoassay instrument, and the sample light absorption value is A
1, blank absorbency is A
2, inhibiting rate (%)=
.The HS that in concentration is 1 mg/mL and 0.25 mg/mL periodate oxidation is 69.8% and 25.80% to the HUVEC inhibiting rate.
Claims (5)
1. the Suleparoid modified of a periodate oxidation.
2. the preparation method of the derivative of claim 1, comprise the following steps:
Get Suleparoid 0.1～2.0 g, swell in 10～40 mL water, add 10～40 mL 0.05～0.5 mol/L NaIO
4, adjust pH is 5.2, fully mixes, and is placed in dark place in 4 ℃ of reaction 24 h, add the ethylene glycol termination reaction,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, Suleparoid after oxidation is dissolved in a small amount of water, under agitation condition, adds approximately 100 mg solid NaBH
4, with the HCl adjust pH, be 3.0, reaction 3 h, then, with the NaOH neutralization,, in 4 ℃ of dialysis 72 h the sample freeze-drying is reclaimed with distilled water, make the Suleparoid of periodate oxidation.
3. by claim 1～2, modify the Suleparoid derivative with inhibition tumor cell and endothelial cell proliferation activity obtain or the single molecule that forms this derivative through periodate oxidation.
4. the single molecule that contains the described Suleparoid derivative of at least a claim 1～3 or form this derivative as activeconstituents and with the pharmaceutical composition of pharmaceutical acceptable carrier and mixed with excipients.
5. the described Suleparoid derivative of claim 1～3 or the single molecule that forms this derivative are for the preparation for the treatment of tumour, the purposes in the medicine relevant with metastases arbitrarily.
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| CN2013103826358A CN103408677A (en) | 2013-08-29 | 2013-08-29 | Periodate-oxidized heparan sulfate for inhibiting cell proliferation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114306746A (en) * | 2021-12-20 | 2022-04-12 | 四川大学 | Preparation method of anticoagulant acellular dermal matrix |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5280016A (en) * | 1991-03-29 | 1994-01-18 | Glycomed Incorporated | Non-anticoagulant heparin derivatives |
-
2013
- 2013-08-29 CN CN2013103826358A patent/CN103408677A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5280016A (en) * | 1991-03-29 | 1994-01-18 | Glycomed Incorporated | Non-anticoagulant heparin derivatives |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114306746A (en) * | 2021-12-20 | 2022-04-12 | 四川大学 | Preparation method of anticoagulant acellular dermal matrix |
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Application publication date: 20131127 |