CN1927415A - Biological material for brain injury renovation and its preparing process - Google Patents
Biological material for brain injury renovation and its preparing process Download PDFInfo
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- CN1927415A CN1927415A CN 200610076650 CN200610076650A CN1927415A CN 1927415 A CN1927415 A CN 1927415A CN 200610076650 CN200610076650 CN 200610076650 CN 200610076650 A CN200610076650 A CN 200610076650A CN 1927415 A CN1927415 A CN 1927415A
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- hyaluronic acid
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- adipic dihydrazide
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Abstract
The invention relates to a biological material used to recover hurt brain. Wherein, it uses transparent hyaluronic acid as raw material, guided by carbodiimide hydrochlorate (EDC), to use adipic acid adipoyl (ADH) to cross, to obtain the hyaluronic acid gel, as the carrier of graft antibody; and uses antibody solidification technique to graft the antibodies of axon growth restrain factor MAG, OMgp and Nogo-A to the hyaluronic acid gel, to be the antibody transfer system carrying special antibody and sensitive to pH value. The invention can be used to seal the never axon regeneration restrain factor of myelin and accelerate the regeneration of hurt brain organism.
Description
Technical field
The present invention relates to a kind of biomaterial that is used for the brain injury reparation and preparation method thereof.
Background technology
Regeneration and Repair behind the brain injury is a difficult point of contemporary medical science.Discover, a reason of cranial nerve regeneration difficulty is, axonal degeneration and myelin disintegrate take place behind the neuronal damage, the Nogo albumen (Nogo-A) that produces after the myelin disintegrate, myelin associated glucoprotein (MAG) and oligodendrocyte myelin glycoprotein materials such as (OMgp) have the effect that suppresses neural axon growth, are called as to suppress the axon growth factor.These suppress the effect that the axon growth factor combines with its receptor NgR in vivo and produces the inhibition neural axon growth.Medical research is also found, combines with NgR with specific antibody blocking-up Nogo-A, MAG and OMgp and just can eliminate the inhibitory action that suppresses axon growth factor pair neuranagenesis, increases the moulding ability of corticospinal tract.Yet how to use this species specific antibodies to become a new technical barrier: the routine administration approach can not pass through blood brain barrier; Heeling-in produces the method for the hybridization tumor cell of antibody in brain, can't effectively control the release of antibody, and has very big tumorigenesis risk; Antibody purified is transported to damage location by heeling-in pump in vivo, can makes the sustainable supply of antibody reach several weeks, but after antibody uses up, need second operation that pump is taken out to the several months.So, contemporary medical science need a kind of effectively and reasonable plan is used for the antibody of NgR the treatment of brain injury.
Summary of the invention
The object of the present invention is to provide a kind of behind brain injury in order to implant the biomaterial of brain essence, it carries the antibody of neural axon growth inhibitive factor receptor, with cerebral tissue better biocompatibility is arranged, its antibody discharges the needs that meet the cerebral tissue Regeneration and Repair.
Adopt tissue engineering technique, (Hyaluronic acid HA) be raw material, and the product that the antibody and the hyaluronic acid of neural axon growth inhibitive factor receptor (NgR) carried out crosslinked gained is the biomaterial that the present invention is used for the brain injury reparation with hyaluronic acid.Described neuritegrowth inhibitor comprises myelin associated glucoprotein, oligodendrocyte myelin glycoprotein and Nogo albumen.Tissue engineering technique involved in the present invention its essence is grafted protein matter molecule on the nonprotein molecule.It is to be raw material with the hyaluronic acid, under the mediation of carbodiimide hydrochloride (EDC), carry out crosslinked method with adipic dihydrazide (ADH) and prepare hyaluronic acid derivatives, carrier material as grafting antibody, be grafted on the hyaluronic acid gel with the antibody of antibody curing technology, make the biomaterial that carries specific antibody the receptor (NgR) of neuritegrowth inhibitor MAG, OMgp and Nogo-A.Concrete grammar is,
1) with deionized water preparation mass percent be 0.8～1.2% hyaluronic acid solution, transferring pH value of solution with hydrochloric acid solution is acidity, and optimum pH value is 4.75; Successively add adipic dihydrazide (ADH) and carbodiimide hydrochloride (EDC), stir.Be 6～8 with the sodium hydroxide solution adjust pH afterwards, get hyaluronic acid gel.Hydrogel is cleaned in ultrasound wave 2～3 times with deionized water, put into the freeze dryer lyophilization after freezing.
2) with the preparation of animal immune method: the antibody of neuritegrowth inhibitor receptor NgR.With step 1. the hyaluronic acid derivatives of method preparation as the carrier material of grafting antibody, be grafted on the hyaluronic acid gel with the antibody of antibody curing technology the receptor (NgR) of neuritegrowth inhibitor MAG, OMgp and Nogo-A, make the biomaterial that carries specific antibody, implant the brain injury position, promote the reparation of cerebral tissue.The hyaluronic acid gel of making by the inventive method that carries antibody is called the antibody induction system.
Wherein, the adding adipic dihydrazide is many more in preparation hyaluronic acid derivatives process, and the hardness of gel is high more.Therefore, with the hardness of the amount control gel that adds adipic dihydrazide, the i.e. hardness of finished product biomaterial.Suitable adipic dihydrazide consumption weight ratio is adipic dihydrazide: hyaluronic acid=1: 4～10; The optimum weight proportioning is adipic dihydrazide: hyaluronic acid=1: 6.Used carbodiimide hydrochloride consumption weight ratio is carbodiimide hydrochloride: hyaluronic acid=1: 2.5～5.0.
Biomaterial by the inventive method making, carry the blocking antibody that suppresses the axon growth factor, have a kind of biochemical characteristic simultaneously: the crack velocity of hydrazone key changes with the variation of environment pH between entrained antibody and the hyaluronic acid, grafting antibody comparatively fast discharges in sour environment, and rate of release slows down under neutral and alkaline environment.Experiment shows, is covalently bonded to the antibody in the hyaluronic acid gel, and in 37 ℃ of environment, in pH was 5.0 solution, 60%～80% antibody discharged in 8 hours; In pH was 6.0 solution, 60%～80% antibody discharged in 70 hours; In pH was 7.4 buffer, antibody discharged quite slow, can continue to discharge 400 hours.
On the other hand, cerebral tissue pH value when damaged can be reduced to 6.2～6.8.When hyperglycemia or hypoxia occurring, pH value can drop to 6.0.The acidosis of cerebral tissue is an of short duration process, generally continues a few minutes to a few hours, then returns to normal biological value.
The above-mentioned characteristic positive adaptation of biomaterial of the present invention the process that pH changes behind the brain tissue impairment, the rate of release of antibody has overcome the disadvantage of antibody " outburst " the formula release of physical method preparation by fast and slow.
The used antibody carrier hyaluronic acid derivatives of biomaterial of the present invention derives from natural polymer, and nontoxic, cell compatibility is good, and is loose porous, is fit to nervous tissue's growth, possesses the rheologic behavio(u)r similar with nervous tissue, can biodegradation, need not to take out.This biomaterial transplantation experiments in cell in vitro cultivation and body confirms to be woven with the good compatibility with neuron cluster, can promote the growing into of regeneration, blood vessel of neural axon, the regeneration that damages back nervous tissue, the recovery of function are had obvious facilitation.
The specific embodiment
The invention will be further described by the following examples.
Embodiment 1 preparation hyaluronic acid gel
With deionized water preparation mass percent is 1% hyaluronic acid solution, and the 1M hydrochloric acid solution is dropwise added hyaluronic acid solution, stirs simultaneously, makes the two fully mixed, reaches 4.75 until its pH value; Take by weighing adipic dihydrazide in " adipic dihydrazide: hyaluronic acid=1: 4～10 " ratio and add in the hyaluronic acid solution, fully stir; Take by weighing carbodiimide hydrochloride in " carbodiimide hydrochloride: hyaluronic acid=1: 2.5～5.0 " ratio again and add in the hyaluronic acid solution, stir 10min～20min; Be 6～8 with the sodium hydroxide solution adjust pH again, get hyaluronic acid gel; Hydrogel is cleaned in ultrasound wave 2～3 times with deionized water, and putting into freeze dryer after freezing carries out lyophilization.
The antibody of embodiment 2 preparation neuritegrowth inhibitor receptor NgR
Check in the full length amino acid sequence of rat NgR in Protein database, sequence analysis shows high antigenic several zones and serves as immune source region.According to antigenicity, specificity and the hydrophilicity of sequence, choosing amino acid residue, to be positioned at 163～176 peptide section DNNLQALPDNTFRD be antigene fragment; Adopt the synthetic DNNLQALPDNTFRD sequence of method of solid phase synthesis, high-efficient liquid phase chromatogram purification, freund adjuvant is crosslinked fully, on a small quantity immunizing rabbit repeatedly.Immunity 6 all rear neck arteries are got blood, separate out serum naturally for 4 ℃, and the centrifugal 20min of 4000r/min obtains the antibody serum of neuritegrowth inhibitor receptor NgR.
The oxidation grafting of embodiment 3 NgR antibody
Sodium metaperiodate is added in the antibody phosphate buffer, and concentration is 10mg/ml, gentle agitation 30 minutes, and dialysis is 3 hours in normal saline, uses the bacteria filter bacteriological filtration then, gets aseptic antibody-solutions; Crosslinked hydrogel is sterilized three times in 75% ethanol, the aseptic phosphate buffer of reuse cleans three times, aseptic antibody is mixed with it, in super-clean bench, reacted 10～20 hours, obtain carrying the hyaluronic acid gel of neuritegrowth inhibitor receptor antibody, be called crosslinked antibody delivery systme.
The check of embodiment 4 hydrogels
Under the mediation of carbon two imide salt hydrochlorates, the carboxyl reaction in hydrazides group in the adipic dihydrazide and the hyaluronic acid in the glycan molecule forms HA-ADH and HA-ADH-HA.Detect to find by infared spectrum, through the hydrogel after crosslinked, respectively 3310 and 3228cm
-1The place has produced two new peaks, and this peak is respectively the flexible and asymmetric flexible bands of a spectrum of symmetry of N-H group.Proof is under the condition of gentleness, and adipic dihydrazide gets final product on the hyaluronic molecular skeleton of covalent cross-linking, and can form free hydrazides group.
Embodiment 5 external antibody release rule are observed
Respectively put the 3ml acetate buffer solution in three cuvettes, their pH value is respectively: pH=5, pH=6, pH=7.2, the trend that pH constantly changes in the brain behind the simulation brain injury.Get three parts of equivalent and insert respectively in three cuvettes by the hyaluronic acid gel that carries antibody of embodiment 3 methods preparation, the measurement of use ultraviolet spectrophotometer is discharged into antibody concentration and the rate of release in the solution.The result shows that pH value is by low and high, and the rate of release of antibody is by fast and slow.
Embodiment 6 antibody activities detect
Through to grafting the hydrogel of antibody carry out external and body in test, detect the activity of the antibody that discharges with immunofluorescence technique, find through keeping the activity of antibody after the grafted antibody release, carry on the back with after the neuronal cell cultures through Embryo Gallus domesticus, find grafting the gel of antibody promote the attaching and the axonal regeneration of neurocyte, the neuroganglion of the antibody capable inducing peripheral of Shi Fanging grows fiber towards the direction of material simultaneously.Tissue regeneration and reconstruction to the rat cerebral ischemia model detect, and confirm that the antibody capable that antibody induction system of the present invention discharges significantly promotes tissue regeneration and functional rehabilitation.
Claims (5)
1. one kind is used for the biomaterial repaired behind the brain injury, it is characterized in that, it is for being raw material with the hyaluronic acid, the product that the antibody and the hyaluronic acid of neuritegrowth inhibitor receptor carried out crosslinked gained; Described neural axon growth inhibitive factor comprises myelin associated glucoprotein, oligodendrocyte myelin glycoprotein and Nogo albumen.
2. the preparation method of the described biomaterial of claim 1 is characterized in that, it comprises following processing step:
1. with deionized water preparation mass percent 0.8～1.2% hyaluronic acid solution, transferring the hyaluronic acid solution pH value with hydrochloric acid solution is acidity, successively adding adipic dihydrazide and carbodiimide hydrochloride, is 6～8 with the sodium hydroxide solution adjust pH again, gets hyaluronic acid gel; Hydrogel is cleaned 2～3 times lyophilization in ultrasound wave with deionized water;
2. will place phosphate buffer with the antibody of the neuritegrowth inhibitor receptor NgR of animal immune method preparation, press 10mg/ml and add sodium metaperiodate, gentle agitation 30 minutes, dialysis is 3 hours in normal saline, use the bacteria filter bacteriological filtration then, get aseptic antibody-solutions; The crosslinked hydrogel that 1. step prepares is sterilized three times in 75% ethanol, and the aseptic phosphate buffer of reuse cleans three times, and aseptic antibody-solutions is mixed with it, reacts in super-clean bench 10～20 hours, obtains crosslinked antibody delivery systme.
3. according to the preparation method of the described biomaterial of claim 2, it is characterized in that transferring the hyaluronic acid solution pH value with hydrochloric acid solution is 4.75.
4. according to the preparation method of the described biomaterial of claim 2, it is characterized in that the consumption weight portion of adipic dihydrazide is adipic dihydrazide in preparation hyaluronic acid derivatives operation: hyaluronic acid=1: 4～10.
5. according to the preparation method of the described biomaterial of claim 2, it is characterized in that the consumption weight portion of adipic dihydrazide is adipic dihydrazide in preparation hyaluronic acid derivatives operation: hyaluronic acid=1: 6.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB200610076650XA CN100536934C (en) | 2006-04-28 | 2006-04-28 | Biological material for brain injury renovation and its preparing process |
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| CNB200610076650XA CN100536934C (en) | 2006-04-28 | 2006-04-28 | Biological material for brain injury renovation and its preparing process |
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| CN1927415A true CN1927415A (en) | 2007-03-14 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657599A (en) * | 2012-05-24 | 2012-09-12 | 哈尔滨工业大学 | Method for preparing bioactive injectable hydrogel materials for oncotherapy |
| CN103013490A (en) * | 2012-12-07 | 2013-04-03 | 深圳康美生物科技股份有限公司 | Method for functionalizing quantum dot biomolecules |
| CN108912353A (en) * | 2018-07-20 | 2018-11-30 | 中南民族大学 | A kind of preparation method and application of sustained-release hydrogel film material |
| CN109481673A (en) * | 2017-09-12 | 2019-03-19 | 中国科学院苏州纳米技术与纳米仿生研究所 | The composition of promotion cerebral injury neural restoration and its application and evaluation method |
| JP2020500833A (en) * | 2016-12-01 | 2020-01-16 | ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッドＲａｍｏｔ Ａｔ Ｔｅｌ Ａｖｉｖ Ｕｎｉｖｅｒｓｉｔｙ Ｌｔｄ． | Combination therapy for nerve damage |
| CN112791229A (en) * | 2021-01-25 | 2021-05-14 | 吉林大学 | A medical dressing containing chlorogenic acid and paeoniflorin and hyaluronic acid hydrogel |
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2006
- 2006-04-28 CN CNB200610076650XA patent/CN100536934C/en not_active Expired - Fee Related
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102657599A (en) * | 2012-05-24 | 2012-09-12 | 哈尔滨工业大学 | Method for preparing bioactive injectable hydrogel materials for oncotherapy |
| CN102657599B (en) * | 2012-05-24 | 2013-09-04 | 哈尔滨工业大学 | Method for preparing bioactive injectable hydrogel materials for oncotherapy |
| CN103013490A (en) * | 2012-12-07 | 2013-04-03 | 深圳康美生物科技股份有限公司 | Method for functionalizing quantum dot biomolecules |
| CN103013490B (en) * | 2012-12-07 | 2016-02-24 | 深圳康美生物科技股份有限公司 | A kind of method of quanta point biological molecules functionalize |
| JP2020500833A (en) * | 2016-12-01 | 2020-01-16 | ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッドＲａｍｏｔ Ａｔ Ｔｅｌ Ａｖｉｖ Ｕｎｉｖｅｒｓｉｔｙ Ｌｔｄ． | Combination therapy for nerve damage |
| JP7271180B2 (en) | 2016-12-01 | 2023-05-11 | ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッド | Combination therapy for nerve damage |
| JP7474894B2 (en) | 2016-12-01 | 2024-04-25 | ラモット・アット・テル・アビブ・ユニバーシテイ・リミテッド | Combination Treatments for Nerve Injury |
| CN109481673A (en) * | 2017-09-12 | 2019-03-19 | 中国科学院苏州纳米技术与纳米仿生研究所 | The composition of promotion cerebral injury neural restoration and its application and evaluation method |
| CN108912353A (en) * | 2018-07-20 | 2018-11-30 | 中南民族大学 | A kind of preparation method and application of sustained-release hydrogel film material |
| CN112791229A (en) * | 2021-01-25 | 2021-05-14 | 吉林大学 | A medical dressing containing chlorogenic acid and paeoniflorin and hyaluronic acid hydrogel |
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| CN100536934C (en) | 2009-09-09 |
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