CN106421805A - Dextran cross-linked hemoglobin-based oxygen carrier, preparation method thereof and application - Google Patents
Dextran cross-linked hemoglobin-based oxygen carrier, preparation method thereof and application Download PDFInfo
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Abstract
The invention discloses a dextran cross-linked hemoglobin-based oxygen carrier, a preparation method thereof and an application. The preparation method includes the steps: oxidizing hydroxy on dextran 40 into an aldehyde group by sodium periodate to obtain activated dextran; protecting sulfydryl on hemoglobin by 4-PDS; crosslinking the activated dextran with the bovine hemoglobin closed by the sulfydryl; finally, de-protecting the sulfydryl to obtain the dextran cross-linked hemoglobin-based oxygen carrier. P50 of the dextran cross-linked hemoglobin-based oxygen carrier (Dex-Hb) prepared by the method can be effectively increased, oxygen carrying-releasing capacity is improved, intramolecular crosslinking of hemoglobin tetramer is realized, so that tetramer stability of HBOCs is remarkably enhanced, and physiological functions of the HBOCs are further improved.
Description
Technical field
The present invention relates to the blood substitute technical field of hemoglobin-based oxygen carrier class, more particularly to a kind of dextran
Cross-linked haematoglobin carrier of oxygen and preparation method and application.
Background technology
Hemoglobin-based oxygen carrier (HBOCs) is by chemical means, hemoglobin to be wrapped up, modified or be crosslinked obtaining
A class material, it is therefore intended that by parcel, modification or crosslinking increase free hemoglobin molecular weight, reduce its nephrotoxicity,
And retain its ability for taking-putting oxygen.But remain in HBOCs research and urgently solve in its development of many restrictions and the problem in science that applies
Certainly.First, easily there is depolymerization in hemoglobin after chemical modification or crosslinking so that the tetramer legibility fusion of hemoglobin is
Dimer；Secondly, modification or crosslinking can also change haemoglobin molecule structure so as to take-put oxygen ability and significantly change.Therefore,
Parcel, modification or the crosslinking reagent that selects and the site for acting on hemoglobin are very big to the performance impact of HBOCs.
As the sulfydryl of Cys-93 (β) on hemoglobin side chain is very active group in hemoglobin.Therefore,
HBOCs majority is the sulfydryl by DCLHb side chain cysteine so as to be connected with macromole dressing agent.Grind
Study carefully proof to be modified when the sulfydryl in hemoglobin, those can be caused to maintain originally T conformation to be converted to the energy of R Conformation Transition
Become the energy for improving oxygen affinity, cause its oxygen affinity to rise, it is difficult to tissue release oxygen so that tissue can not be obtained
Enough oxygen, exacerbates the anoxia-induced apoptosis of tissue.How preferably the stabilizing hemoglobin tetramer, improve which and take-put oxygen ability
It is the key issue for needing in HBOCs research to solve.
Dextran crosslinking is a kind of technological means for preparing HBOCs, conventional sodium periodate oxidation.Sodium periodate oxidation
Method is as oxidant using sodium metaperiodate, the hydroxyl oxygen on dextran is melted into aldehyde radical, afterwards the ammonia with haemoglobin molecule
Base carries out cross-linking reaction.However, the sodium metaperiodate after oxidized dextran in reactant liquor can not possibly be eliminated completely.Due to periodic acid
Sodium is a kind of strong oxidizer, and the sodium metaperiodate of residual and the oxidized dextran containing multiple aldehyde radicals can aoxidize blood red egg
Sulfydryl on white side chain so that haemachrome microenvironment changes, and causes dextran cross-linked haematoglobin (Dex-Hb) oxygen affinity
Too high with power, take-put the reduction of oxygen ability, it is impossible to play oxygen supply effect well, therefore cannot function as blood substitute use.
Content of the invention
The purpose of the present invention be for technological deficiency present in prior art, in a first aspect, provide one kind can stablize
Tetrameric hemoglobin body, and the dextran cross-linked haematoglobin carrier of oxygen for taking-putting oxygen ability can be improved, by dextran and blood
Lactoferrin crosslinking is obtained, and includes two sulfydryls in the carrier of oxygen on each tetrameric hemoglobin body.
Second aspect, the present invention provides a kind of method for preparing the above-mentioned dextran cross-linked haematoglobin carrier of oxygen, by blood
Mix with activated dextran acid anhydride after sulfhydryl protected on Lactoferrin, crosslink reaction；Sulfydryl is carried out after the completion of crosslinking again
Deprotection, obtains the dextran cross-linked haematoglobin carrier of oxygen.
Comprise the following steps：
1) with sodium metaperiodate, the hydroxyl oxygen on Dextran 40 is melted into aldehyde radical, obtains activated dextran acid anhydride；
2) use 4,4 '-two sulfur, two pyridine will be sulfhydryl protected in hemoglobin, obtain the blood red egg after sulfydryl closing
In vain；
3) by step 1) activated dextran acid anhydride and step 2) sulfydryl closing after hemoglobin mix, carry out being crosslinked instead
Should, obtain the dextran cross-linked haematoglobin carrier of oxygen of sulfydryl closing；
4) by step 3) sulfydryl closing the dextran cross-linked haematoglobin carrier of oxygen carry out sulfydryl deprotection, obtain the right side
The sugared acid anhydride cross-linked haematoglobin carrier of oxygen of rotation, includes two sulfydryls on each tetrameric hemoglobin body in the carrier of oxygen.
Step 2) it is specially：
Anhydrous alcohol solution is used after weighing 4,4 '-two sulfur, two pyridine, be added thereto to hemoglobin and 4 times of dehydrated alcohol bodies
The buffer B of product, mix homogeneously is reacted under room temperature condition, obtains the hemoglobin after sulfydryl closing；Hemoglobin and 4,
The mol ratio of 4 '-two sulfur, two pyridine is 1:(4-8) (preferably 1：(4-6), most preferably 1:4).
Step 2) response time be 3 hours；Preferably, the buffer B is 20mM phosphate buffer (pH7.4).
Step 1) it is specially：
Weigh Dextran 40 respectively to be dissolved in buffer A with sodium metaperiodate so that Dextran 40 final concentration of
2.5mg/ml, the final concentration of 10mM of sodium metaperiodate, obtain reaction system, and room temperature lucifuge is reacted 90 minutes, by Dextran 40
On hydroxyl oxygen chemical conversion aldehyde radical；Reaction adds ethylene glycol standing 1-3h after terminating, terminate sodium periodate oxidation reaction, and rate of charge is
1mg sodium metaperiodate adds 3-6 μ l ethylene glycol；Dialysed with NaCl solution and buffer B at room temperature after reaction terminating respectively, remove
The sodium metaperiodate of residual and ethylene glycol (using the bag filter of 10kDa), obtain activated dextran acid anhydride；Preferably, the buffer A
Acetate-acetate buffer solution (pH5.8) for 20mM sodium acetate.
Step 3) it is specially：
By step 2) hemoglobin after the closing of the sulfydryl that obtains, step 1) the activated dextran acid anhydride that obtains and cyano group boron hydrogen
Change sodium powder end in mass ratio 1:(0.05-5):(0.25-2.5) mix homogeneously (preferably 1:(1-2):(0.5-1), most preferably 1:1:
0.5), hemoglobin and dextran crosslinking are overnight got up by lucifuge reaction；Ultrafiltration is carried out with buffer B after reaction completely, obtain
The dextran cross-linked haematoglobin carrier of oxygen to sulfydryl closing.
Step 4) it is specially：
Three (2- carboxyethyl) phosphonium salt hydrochlorate is weighed, with as deprotection agent after pure water dissolving, three (2- carboxyethyl) phosphonium salt is sour
Salt is 4 times of hemoglobin molal quantity, to step 3) the dextran cross-linked haematoglobin carrier of oxygen of sulfydryl closing that obtains surpasses
Filter, carries out deprotection to the sulfydryl that closes；Continuation buffer B ultrafiltration after reaction completely, obtains dextran and is crosslinked blood red egg
The white carrier of oxygen.
The third aspect, the present invention provides a kind of dextran cross-linked haematoglobin carrier of oxygen, is prepared into by said method
Arrive.
Fourth aspect, the present invention provides the above-mentioned dextran cross-linked haematoglobin carrier of oxygen answering in blood substitute
With the carrier of oxygen being applied in transfusion procedure (wound, lose blood, anemia, operation etc. cause), is partly or entirely substituted red
Oxygen function is taken/put to cells play, and its oxygen saturation is partial pressure of oxygen P when 50%50In more than 9.00mmHg.
Compared with prior art, the invention has the beneficial effects as follows：
The present invention is improved to the method for dextran cross-linked haematoglobin, is preparing dextran cross-linked haematoglobin
Reversible protection HbC ys-93 (β) sulfydryl during the carrier of oxygen (Dex-Hb) so as to not oxidized.As a result confirm：This
The P of the dextran cross-linked haematoglobin carrier of oxygen (Dex-Hb) that the method for invention is obtained50Can effectively improve, which takes-put oxygen
Ability is also improved.As dextran is aoxidized on the multiple aldehyde groups for producing energy four subunits simultaneously with hemoglobin
Amino reaction, realize the intramolecular crosslinking of tetrameric hemoglobin body, so as to dramatically increase the tetramer stability of HBOCs, enter
And improve the physiological function of HBOCs.
Description of the drawings
Fig. 1 (A) width represents 3 gel filtration chromatography figure of embodiment, and (B) width represents the experimental result of SDS-PAGE；
Fig. 2 show the oxygen dissociation curve figure of embodiment 3；
Fig. 3 (A) width represents the ferrihemoglobin content figure of embodiment 3, and (B) width represents gel filtration chromatography figure；
Fig. 4 show the extent of reaction figure of embodiment 3, embodiment 4, comparative example 1 and comparative example 2.
Specific embodiment
The preparation of buffer：
1. Acetate-acetate buffer solution (NaAC-HAC, pH5.8,20mM, hereinafter referred to as " buffer A ")
Anhydrous sodium acetate 1.64g being weighed, is dissolved in 900ml pure water, pH to 5.8 is adjusted with glacial acetic acid, be settled to pure water
1L, obtains final product.
2. phosphate buffer (PB, pH7.4,20mM, hereinafter referred to as " buffer B ")
4.8g AMSP (119.98) and 14.8g disodium hydrogen phosphate (358.14) is weighed, is dissolved in 4L
Dissolve in pure water, obtain final product.
The method for preparing the dextran cross-linked haematoglobin carrier of oxygen of the present invention, comprises the following steps：
1) weigh Dextran 40 respectively to be dissolved in buffer A with sodium metaperiodate (weighing under the conditions of lucifuge), obtain anti-
Answer system so that the final concentration of 2.5mg/ml of Dextran 40, the final concentration of 10mM of sodium metaperiodate, reaction system room temperature is kept away
Photoreaction 90min, the hydroxyl oxygen on Dextran 40 is melted into aldehyde radical by sodium metaperiodate.Second is added in the reaction system after oxidation
Glycol, stands 1-3h, terminates sodium periodate oxidation reaction, and rate of charge adds 3-6 μ l ethylene glycol for 1mg sodium metaperiodate.To terminate
The reaction system of oxidation is dialysed twice with the NaCl solution room temperature of 0.15M, each 1h, then is dialysed 1 time with buffer B room temperature, when
Between be 3h, remove residual sodium metaperiodate and ethylene glycol (using the bag filter of 10kDa), obtain activated dextran acid anhydride.
2) it is 4,4 '-two sulfur, two pyridines (4-PDS) in molar ratio:Bovine hemoglobin=(4-8):1 rate of charge weighs 4-
PDS, with anhydrous alcohol solution 4-PDS (after the volume (mL) of dehydrated alcohol is 20-30 times of 4-PDS mass (g), thereto plus
Enter buffer B (volume is 4 times of dehydrated alcohol)), adding bovine hemoglobin mix homogeneously carries out closing the reaction of sulfydryl, room
Temperature reaction 2-5h, obtains the bovine hemoglobin after sulfydryl closing.
3) by step 2) bovine hemoglobin after the closing of the sulfydryl that obtains, step 1) the activated dextran acid anhydride that obtains and cyano group
Sodium borohydride powder in mass ratio 1:(0.05-5):(0.25-2.5) mix (preferably 1:(1-2):(0.5-1), most preferably 1:1:
0.5), react overnight (8-12h) in 4 DEG C of lucifuges, obtain the dextran cross-linked haematoglobin carrier of oxygen containing sulfydryl closing
Reactant liquor.By step 3) reactant liquor carry out ultrafiltration with buffer B, set flow velocity as 4.0ml/min, ultrafiltration 7-9h, obtain mercapto
The dextran cross-linked haematoglobin carrier of oxygen of base closing.
4) it is bovine hemoglobin in molar ratio:Three (2- carboxyethyl) phosphonium salt hydrochlorate (TCEP HCl)=1:4 rate of charge claims
TCEP HCl is taken, after pure water dissolving, as deprotection agent, to step 3) the dextran crosslinking blood of the sulfydryl that obtains closing
The Lactoferrin carrier of oxygen proceeds ultrafiltration, releases the closing to sulfydryl, reacts 40min.The buffer B ultrafiltration 7- of continuation afterwards
8h, obtains the dextran cross-linked haematoglobin carrier of oxygen of liquid.
5) sample, -80 DEG C of preservations are collected.
Below in conjunction with specific embodiment, present disclosure is further illustrated, and the present invention is further elaborated, but
These embodiments are limited the invention absolutely not.
Embodiment：
A series of dextran cross-linked haematoglobin carriers of oxygen are prepared in aforementioned manners, simply the parameter of preparation process
Slightly adjust, remaining program is constant, the parameter of preparation process is shown in Table 1.
1 present invention of table prepares the parameter of the method for the dextran cross-linked haematoglobin carrier of oxygen
Comparative example 1：By taking embodiment 3 as an example, the present invention is prepared the dextran cross-linked haematoglobin carrier of oxygen of the present invention
Step 3 in method) mass ratio of bovine hemoglobin, activated dextran acid anhydride and sodium cyanoborohydride powder after sulfydryl closing is changed to
1: 0.01: 0.5, other steps and parameter constant, as a result see Fig. 4.As a result show：The cross-linking effect of embodiment 3 and embodiment 4 is relatively
Good, remaining hemoglobin content is less, it is considered to economy principle, and reaction is than, during for 1: 1: 0.5, can more save the original of cross-linking reaction
Material；And responseless hemoglobin is more in comparative example 1, product purity is poor.
Comparative example 2：By taking embodiment 3 as an example, the present invention is prepared the dextran cross-linked haematoglobin carrier of oxygen of the present invention
Step 3 in method) mass ratio of bovine hemoglobin, activated dextran acid anhydride and sodium cyanoborohydride powder after sulfydryl closing is changed to
1：0.5:0.1, other steps and parameter constant, as a result see Fig. 4.As a result show：Responseless hemoglobin is more, and product is pure
Degree is poor.
Experiment one：SDS-PAGE (SDS- polyacrylamide gel electrophoresis) and gel filtration chromatography
Press《Molecular Cloning:A Laboratory guide》The SDS- polyacrylamide gel of the protein of page 1713 in (third edition) volume two
Method in electrophoresis carries out SDS-PAGE experiment to embodiment 1-8.After preparing 12% separation gel and 5% concentration glue, electricity is installed
Swimming system, adds electrophoretic buffer；Will be (blood red for the dextran cross-linked haematoglobin oxygen carrier sample of embodiment of the present invention 1-8
Protein content is 35mg) with the sample-loading buffer (loading buffer) of 5 times of volumes (i.e.+20 μ l 5 of 5 μ l sample ×
Loading buffer) mixing；Heating 5min in boiling water is put into, is centrifuged, takes 20 μ l supernatant loadings；Using Bole's electrophresis apparatuses, surely
Pressure 120V 30min, makes sample enter separation gel, and voltage stabilizing 90V runs electrophoresis afterwards；Dyeing, decolouring, record experimental result.
By Wang et al.Biochim Biophys Acta 2014,1844:Experimental technique in 1201-1207 is to reality
Applying a 1-8 carries out the experiment of gel filtration chromatography.
By taking embodiment 3 as an example, in Fig. 1, (A) width represents the result of gel filtration chromatography, and (B) width represents the knot of SDS-PAGE
Really.
From Fig. 1, the SDS-PAGE electrophoresis result of (B) width can be seen that hemoglobin (Hb) and only show at 16kDa
One band, does not have other bands to occur.Demonstrate Hb purity higher, meet preparation and require.Dextran cross-linked haematoglobin
The carrier of oxygen (Dex-Hb) only shows a band in more than 100kDa, illustrates that the purity of Dex-Hb is higher.And 97kDa with
Under, Dex-Hb does not have band to occur, and the molecular weight of Dex-Hb is described more than 97kDa, and cross-linking reaction significantly increases hemoglobin
The molecular weight of molecule.
From Fig. 1 the gel filtration chromatography figure of (A) width can be seen that Hb occur in that one symmetrical unimodal, illustrate Hb's
Purity is higher, occurs without impurity.Following conclusion can be obtained from the appearance time of Dex-Hb and peak type：(1) Dex-Hb only goes out
Showed one symmetrically unimodal, illustrate that Dex-Hb purity is higher, without other impurities.(2) appearance time of Dex-Hb is said early than Hb
The molecular weight of bright Dex-Hb is more than Hb, and cross-linking reaction has successfully increased the molecular weight of haemoglobin molecule.
Other embodiments also have similar effect, no longer repeat one by one.
Experiment two：Take-put oxygen ability
By the dextran cross-linked haematoglobin carrier of oxygen (6mg containing hemoglobin) of embodiment 1-8 and the P of 4ml pH7.450
Buffer (P50Buffer：7.89g Sodium Chloride, 6.89g TES and 0.373g potassium chloride is weighed, is dissolved in ultra-pure water, at 37 DEG C
PH=7.4 ± 0.02 is adjusted, osmotic pressure is 295 ± 10mOsm/kg, 1L to be settled to, and 4 DEG C save backup) mix, control reactant
The temperature of system is 37 DEG C, with blood oxygen analysis instrument determine reaction system oxygen dissociation curve (by taking embodiment 3 as an example, the song seen in Fig. 2
Line b) and P50(oxygen saturation is partial pressure of oxygen when 50%).The oxygen dissociation curve of hemoglobin (Hb) is obtained using same method
(see the curve a) in Fig. 2 and P50).
P50Be oxygen saturation be partial pressure of oxygen when 50%, be to characterize the important indicator that oxygen ability was taken-put to hemoglobin.Fig. 2
Represent by inventive closure sulfydryl method crosslinking obtain product b (i.e. embodiment 3 dextran cross-linked haematoglobin oxygen carry
Body) it is crosslinked oxygen dissociation curve and the P of the product a for obtaining with unclosed sulfydryl50Situation of change.Unclosed mercapto as can be seen from Figure 2
Dex-Hb (the P of product a) of base50Too low, it is 4.61mmHg, P50Too low be unfavorable for tissue provide oxygen.With unclosed sulfydryl
Dex-Hb (product a) is compared, and (product b) oxygen dissociation curve is moved to right, and P for obtained Dex-Hb after closing sulfydryl50Increase to
9.86mmHg.Compared with the Dex-Hb of unclosed sulfydryl, the P of the Dex-Hb of sulfydryl is closed50Significantly improve, it was demonstrated that closing sulfydryl side
What method can be effectively improved Dex-Hb takes-puts oxygen ability.
Other embodiments also have similar effect, no longer repeat one by one.
Experiment three：Tetrameric hemoglobin body stability
According to Benesch RE, Benesch R, Yung S.Equations for the spectrophotometric
analysis of hemoglobin mixtures.Anal Biochem,1973,55(1)：245-248. in method to this
Inventive embodiment 1-8 carries out metahemoglobin detection, specially：Preparation PBS solution, 0.22 μm of membrane filtration, standby；Point
The dextran cross-linked haematoglobin carrier of oxygen (Dx40-Hb) that other Example 1-8 is obtained and Hb as sample load 15ml from
In heart pipe, constant volume to 10ml, it is ensured that the final concentration of 1.6mg/ml of hemoglobin；MgCl is separately added into in sample2To final concentration
For 0.9M；Take 1ml sample respectively, survey its absorbance in 540nm, 560nm, 576nm, 630nm, 700nm；By remaining sample
MgCl is not added with (2Sample) be put in water-bath, 37 DEG C of water-baths, respectively in 1h, 2h, 3h, 4h, 6h, 7h, take 1ml solution, survey
Which is in the absorbance of 540nm, 560nm, 576nm, 630nm, 700nm；0h, 1h, 2h, 3h, 4h, 6h, 7h are calculated with Spectrophotometry Method Using Three-wavelength
Metahemoglobin percentage composition；Mapping, by taking embodiment 3 as an example, is as a result shown in Fig. 3 (A).
Gel filtration chromatography experimentation (identical with one method of experiment)：Take Hb sample respectively and Dex-Hb sample is (blood red
Protein content is 300 μ g) in 150 μ l PBS, magnesium chloride is added to final concentration 0.9M, 300 μ l of total system, 37 DEG C of reactions
150min, crosses 0.45 μm of filter, takes 100 μ l loadings；Setting Detection wavelength is 280nm, and loading speed is 0.5mg/ml, obtains solidifying
Glue filtering chromatogram figure, by taking embodiment 3 as an example, is as a result shown in Fig. 3 (B).
Fig. 3 represents closing impact of the sulfydryl to tetrameric hemoglobin body stability.MgCl2Hemoglobin can be promoted by four
Aggressiveness depolymerization is changed into dimer, so as to easily aoxidize, when hemoglobin oxygen turns to metahemoglobin, loses and combines oxygen
Function, that is, lose biological action, thus when hemoglobin-based oxygen carrier is prepared require ferrihemoglobin content lower
Better.Fig. 3 (A) shows in MgCl2Under effect, substantially aoxidizing occurs in Hb, variable quantity nearly 100% (curve a), and for Dex-
For Hb, its MetHb changes of contents is little, and variable quantity is less than 30% (curve b), it was demonstrated that the Dex-Hb tetramer is far above Hb；Bent
Line c and d essentially coincide the change for showing that the inventive method does not cause hemoglobin oxidation state in cross-linking process, therefore
To Dex-Hb without MgCl2In the case of, basically identical with the tetramer structure stability of hemoglobin.
Gel filtration chromatography result shows Fig. 3 (B) in MgCl2In the presence of, Hb appearance time is delayed, and in elution volume
There are two peaks (as shown by arrows in FIG.) before and after 20ml, illustrate that obvious depolymerization occurs in Hb；And Dex-Hb is in MgCl2Go out under effect
Peak time is basically unchanged, and only presents unimodal, illustrates that its depolymerization is not obvious；The result confirms：Closing sulfydryl Dex-Hb has relatively
High tetramer stability.
Other embodiments also have similar effect, no longer repeat one by one.
The above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also present disclosure should be regarded as.
Claims (10)
1. a kind of dextran cross-linked haematoglobin carrier of oxygen, is obtained with hemoglobin crosslinking by dextran, it is characterised in that
Include two sulfydryls in the carrier of oxygen on each tetrameric hemoglobin body.
2. a kind of method for preparing the dextran cross-linked haematoglobin carrier of oxygen described in claim 1, it is characterised in that will be blood red
Mix with activated dextran acid anhydride after sulfhydryl protected on albumen, crosslink reaction；Carry out sulfydryl after the completion of crosslinking again to take off
Protection, obtains the dextran cross-linked haematoglobin carrier of oxygen.
3. method according to claim 2, it is characterised in that comprise the following steps：
1) with sodium metaperiodate, the hydroxyl oxygen on Dextran 40 is melted into aldehyde radical, obtains activated dextran acid anhydride；
2) use 4,4 '-two sulfur, two pyridine will be sulfhydryl protected in hemoglobin, obtain the hemoglobin after sulfydryl closing；
3) by step 1) activated dextran acid anhydride and step 2) sulfydryl closing after hemoglobin mix, carry out cross-linking reaction,
Obtain the dextran cross-linked haematoglobin carrier of oxygen of sulfydryl closing；
4) by step 3) sulfydryl closing the dextran cross-linked haematoglobin carrier of oxygen carry out sulfydryl deprotection, obtain dextrose
The acid anhydride cross-linked haematoglobin carrier of oxygen, includes two sulfydryls on each tetrameric hemoglobin body in the carrier of oxygen.
4. method according to claim 3, it is characterised in that step 2) it is specially：
Weigh after 4,4 '-two sulfur, two pyridine with anhydrous alcohol solution, be added thereto to hemoglobin and 4 times of dehydrated alcohol volumes
Buffer B, mix homogeneously is reacted under room temperature condition, obtains the hemoglobin after sulfydryl closing；Hemoglobin and 4,4 '-two
The mol ratio of two pyridine of sulfur is 1:(4-8) (preferably 1：(4-6), most preferably 1:4).
5. method according to claim 4, it is characterised in that step 2) response time be 3 hours；Preferably, described slow
Liquid B is rushed for 20mM phosphate buffer (pH 7.4).
6. according to the arbitrary methods described of claim 3-5, it is characterised in that step 1) it is specially：
Weigh Dextran 40 respectively to be dissolved in buffer A with sodium metaperiodate so that Dextran 40 final concentration of
2.5mg/ml, the final concentration of 10mM of sodium metaperiodate, obtain reaction system, and room temperature lucifuge is reacted 90 minutes, by Dextran 40
On hydroxyl oxygen chemical conversion aldehyde radical；Reaction adds ethylene glycol standing 1-3h after terminating, terminate sodium periodate oxidation reaction, and rate of charge is
1mg sodium metaperiodate adds 3-6 μ l ethylene glycol；Dialysed with NaCl solution and buffer B at room temperature after reaction terminating respectively, remove
The sodium metaperiodate of residual and ethylene glycol (using the bag filter of 10kDa), obtain activated dextran acid anhydride；Preferably, the buffer A
Acetate-acetate buffer solution (pH5.8) for 20mM sodium acetate.
7. according to the arbitrary methods described of claim 3-6, it is characterised in that step 3) it is specially：
By step 2) hemoglobin after the closing of the sulfydryl that obtains, step 1) the activated dextran acid anhydride that obtains and sodium cyanoborohydride
Powder in mass ratio 1:(0.05-5):(0.25-2.5) mix homogeneously (preferably 1:(1-2):(0.5-1), most preferably 1:1:0.5),
Hemoglobin and dextran crosslinking are overnight got up by lucifuge reaction；Ultrafiltration is carried out with buffer B after reaction completely, obtain sulfydryl
The dextran cross-linked haematoglobin carrier of oxygen of closing.
8. according to the arbitrary methods described of claim 3-7, it is characterised in that step 4) it is specially：
Three (2- carboxyethyl) phosphonium salt hydrochlorate is weighed, with as deprotection agent after pure water dissolving, three (2- carboxyethyl) phosphonium salt hydrochlorate is
4 times of hemoglobin molal quantity, to step 3) the dextran cross-linked haematoglobin carrier of oxygen ultrafiltration of the sulfydryl that obtains closing, right
The sulfydryl of closing carries out deprotection；Continuation buffer B ultrafiltration after reaction completely, obtains dextran cross-linked haematoglobin oxygen load
Body.
9. a kind of dextran cross-linked haematoglobin carrier of oxygen, it is characterised in that be by the arbitrary methods described system of claim 2-8
Standby obtain.
10. the dextran cross-linked haematoglobin carrier of oxygen described in claim 1 or the arbitrary methods described of claim 2-8 are prepared into
To application of the dextran cross-linked haematoglobin carrier of oxygen in blood substitute, the carrier of oxygen is applied in transfusion procedure
In (wound, lose blood, anemia, operation etc. cause), partly or entirely substitute erythrocyte and play and takes/put oxygen function, its oxygen saturation
Spend for partial pressure of oxygen P when 50%50In more than 9.00mmHg.
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| CN112129946A (en) * | 2020-08-16 | 2020-12-25 | 陆修委 | Preparation method and application of sugar-free chain type inert protein sealant |
| CN114617957A (en) * | 2022-03-10 | 2022-06-14 | 中国人民解放军军事科学院军事医学研究院 | Hydroxyethyl starch hemoglobin conjugate and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112129946A (en) * | 2020-08-16 | 2020-12-25 | 陆修委 | Preparation method and application of sugar-free chain type inert protein sealant |
| CN114617957A (en) * | 2022-03-10 | 2022-06-14 | 中国人民解放军军事科学院军事医学研究院 | Hydroxyethyl starch hemoglobin conjugate and preparation method and application thereof |
| CN114617957B (en) * | 2022-03-10 | 2024-01-16 | 中国人民解放军军事科学院军事医学研究院 | Hydroxyethyl starch hemoglobin conjugate, and preparation method and application thereof |
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| CN106421805B (en) | 2019-06-04 |
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