CN1161457C - In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material - Google Patents
In vitro reconstituted human erythrocyte, its preparation and application in blood substitute material Download PDFInfo
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Abstract
The present invention relates to human red blood cells reconstructed in vitro, the preparation thereof, and the application of the human red blood cells in blood substitutes. The human red blood cells reconstructed in vitro are constructed by haemoglobin, structural protein and phospholipid and have the morphology of a double concave disk, which is similar to the morphology of natural human red blood cells. The physiological functions and the morphology of the red blood cells reconstructed in vitro of the present invention are close to the physiological functions and the morphology of natural red blood cells, the red blood cells of the present invention have favorable deformability and stability, the strong capability of oxygen carrying and oxygen release, and immunization removal, and the cell size can be controlled to the nanometer level. The present invention also relates to a preparing method for the reconstructed red blood cells and the application of the reconstructed red blood cells in blood substitutes.
Description
Technical field
The present invention relates to a kind of by reconstruction in vitro artificial red blood cellses such as structural protein (erythrocytic membranin or soybean 11S sphaeroprotein), phosphatide, oxyphorases, as novel blood substitute.To be that basic molecular material is collaborative with structural protein, phosphatide seal oxyphorase in the present invention, the reconstruction in vitro artificial red blood cells; By combining of oxyphorase and structural protein, phosphatide, reconstruction in vitro goes out physiological function and form near the neutral red cell, have deformability, good stability, take oxygen-the oxygen release ability is strong, go immunization, and the cell size can control to nano level reconstruction red corpuscle.The present invention relates to a kind of by reconstruction in vitro artificial red blood cellses such as structural protein, phosphatide, oxyphorases, as novel blood substitute.
Background technology:
Up to now, medically used blood substitute all can only be on partial function and structure normoerythrocyte in the analogue body, and ignored the material impact of red corpuscle biomechanics characteristic to its physiological function.Discover, have inseparable inner link between erythrocytic mechanical characteristic and its biochemical functions.Normal neutral red cell is except having the oxygen of taking and energy metabolism function, and its composition structure is given red corpuscle and had specific concave-concave disc form, good changeable type, makes it and can successfully successfully circulate in the blood vessel of various calibers in vivo.The red corpuscle of reconstruction in vitro not only will possess normal biochemical functions, and to possess good deformability, studies show that: erythrocytic cytolemma material--structural protein are crucial in the effect of keeping the specific concave-concave disc form of red corpuscle, guarantee the red corpuscle normal physiological function and giving aspects such as erythrocyte deformability.
At present for the demand of safe and effective blood substitute also in continuous increase, in clinical medicine practise, use blood to be subjected to the restriction of strictness during transfusion.Mainly cause owing to erythrocytic natural characteristic and risk of disease transmission.There are a series of problems in traditional personnel's blood transfusion mode: 1. natural blood storage and transportation are also very difficult: blood storage time is short, and the longest shelf time is 42 days under 4 ℃ of-8 ℃ of conditions, if long-distance transport also easily causes haemolysis.2. because it exists various viruses to comprise the danger that acquired immune deficiency syndrome (AIDS), hepatitis etc. are contaminated, though the blood donor screens through strict blood count, the danger of hepatitis C is still 1: 3000, it is 1: 100000～1: 1000000 that HIV infects; Blood transfusion also can cause other viruses, bacterium, parasite and other transmissions of disease.3. blood transfusion needs to join type for acceptor, otherwise immune response occurs, has the compatibility of different blood groups, the problems such as shortage of special blood group.4. the aging of population, healthy blood supply crowd deficiency, fresh blood famine blood source, certain areas is problem such as exhausted wretched insufficiency relatively, brings serious difficulty and challenge for clinical treatment, first aid; Particularly in war, serious natural disaster, terror accident, only depend on blood bank's blood supply, personnel to donate blood and be difficult to practical requirement especially, therefore to blood substitute particularly on performance and the function demand market of the most approaching normocytic " reconstruction red corpuscle " be difficult to the appraisal.
For a long time, the research of blood substitute is the goal in research of preclinical medicine, clinical medicine and related discipline always.Originally blood substitute is mainly with replenishment of blood content, remedy because the nutritive substance that causes of losing blood is lost and be purpose, nutrition fluid infusion by compatibility replenishes the blood plasma that loses, but this can not give human body delivery of oxygen timely, can't correct the hypoxic damage of the back human body of losing blood; The appearance of can rich long-pending oxygen and can the organic application identity of fluoro containing polymers of slowly-releasing oxygen novel blood substitute in blood, newer blood substitute is with ox at present, the sheep oxyphorase is after removing its immunological characteristic, form macromole and immobilization on inert support by crosslinked (cross-linking) between the molecule, or the expressing gene of oxyphorase is transferred in the plant by transgenic technology, obtain oxyphorase by plant, in blood circulation in the simulation normoerythrocyte natural hemoglobin take oxygen-oxygen release physiological function, but this mimic blood substitute is not owing to there is the barrier of cytolemma, easily and the many bioactive molecule effects in the blood, bring out a series of unusual side reactions, at present used blood quid pro quo all contains oxyphorase (hemoglobin), all can occur the situation of blood pressure increase (increased blood pressure) when a lot of with the back patient.This is owing to be used for keeping normotensive NO, reacts with oxyphorase, and NO is depleted, so increased blood pressure.At present the best blood substitute of biocompatibility should belong to the artificial red blood cells: promptly with liposome membrane parcel natural hemoglobin molecule, or gene recombination oxyphorase and chemosynthesis oxyphorase become artificial red blood corpuscle, and size is similar to red blood corpuscle.But the problem of its existence is: it is not high that this lipoid plastid artificial red blood cells takes oxygen efficiency, and film is easily crisp, and oxyphorase leaks, and the shelf-time is short to tens minutes, long several days only.
The ideal blood substitute is to have the advantage of normal blood and overcome its insufficient product.The ideal blood substitute should possess: 1. high oxygen carrying capacity, have good rheological property, can improve the promotion microcirculation; 2. work as oxygen tension and in the normal physiological scope, can transport oxygen; 3. the removing characteristic that has hope; 4. have no side effect; 5 storage periods long (life-span of blood substitute very short, short then several hours, MaLS is more than 70 hours, the natural erythrocytic life-span then is 120 days) at present, do not have join the type needs, transfusion reaction reaches and does not activate normal function of immune system.A kind of blood substitute that also do not have of development has whole advantages of blood at present.
The reason of blood substitute above shortcomings is at present: existing blood substitute all lays particular emphasis on normocytic biochemical functions of simulation such as function of carrying oxygen, and most researchers is not recognized the importance of simulation normoerythrocyte mechanical characteristic and rheological properties.The neutral red cell mainly organically is made of oxyphorase, membrane phospholipid and structural protein, between oxyphorase and structural protein, the film fat close structural dependence is arranged.The structural protein of keeping the cell specific modality, the channel protein that also has responsible oxygen to transmit in forming, structural protein are arranged; Oxyphorase is that oxyphorase anchors on the film with structural protein by combining, and is that the conformational change of oxyphorase helps in conjunction with oxygen; Structural protein form the passage that helps the oxygen transmission most with film fat again by hydrophobic interaction.This just mutual structural relation makes red corpuscle possess good deformability and visco-elasticity and takes oxygen-oxygen release function normally.These structural relations should be can not ignore in framework reconstruction artificial red blood cells's process.The reason that present liposomal encapsulated stabilized hemoglobin and function of carrying oxygen do not reach requirement is, research has in the past been ignored between oxyphorase and the structural protein, the structural relation between structural protein and the film fat, and this relation is sealed and only with film fat oxyphorase is carried out physical property the contribution that red corpuscle has good changeable type and rheological property.
The content of invention
Influence the structural protein of red cell deformability in analysis and research, on the basis of work such as film fat molecule structure change mechanism, present the blood substitute particularly structure of liposomal encapsulated oxyphorase and the shortcoming of existence have been analyzed, one of technical problem to be solved by this invention is: propose to form normocytic three basic molecular materials--oxyphorase, ad hoc structure albumen, membrane phospholipid is the basis, use phosphatide, the collaborative oxyphorase of sealing of structural protein goes out physiological function and the approaching natural erythrocytic artificial red blood cells of form at reconstruction in vitro, and based on this, constitute the best blood substitute of biocompatibility.Described artificial red corpuscle is because by oxyphorase and structural protein, the organic keying action of membrane phospholipid, mechanical characteristics such as its deformability, fragility and film visco-elasticity near the neutral red cell, have normoerythrocyte concave-concave butterfly dish type attitude, stability is taken characteristics such as oxygen-oxygen release ability is strong.
Two of technical problem to be solved by this invention provides the method for the HRBC of the above-mentioned reconstruction in vitro of preparation.
Three of technical problem to be solved by this invention is: above-mentioned reconstruction in vitro is gone out physiological function and form be used to prepare blood substitute near natural erythrocytic HRBC.
For one of solving the problems of the technologies described above, the technical solution used in the present invention is such: promptly a kind of phosphatide-structural protein are collaborative seals oxyphorase, and the artificial red blood cells of external structure comprises following material:
1. oxyphorase: come from blood productss such as people, ox, sheep, pig; Or by making up gene, the product that vector expression such as intestinal bacteria or yeast or secretion obtain; Or by transgenic technology, the product that obtains by plant vector production.
2. structural protein: for coming from the membranin of people, ox, sheep, pig erythrocyte; Come from the protein product that natural plant protein product such as glycinin 11S have similar function.Its molecular weight: 250,000～620,000 dalton; Its ultra-violet absorption spectrum is: λ
Max=280nm (physiological saline); High-visible three protein protomer absorption peaks in gel electrophoresis UV scanning collection of illustrative plates, wherein to account for the per-cent of total protein peak area be 15%～26%, 12%～24%, 15%～20% to the peak area at each peak.
3. phosphatide: come from the natural phant concentrated phosphatide, obtain product through separation and purification; Or the phosphatide product that obtains by microbial fermentation technology.
In the mentioned component, structural protein and phosphatide constitute erythrocytic cell membrane component, and in cell membrane component, by weight percentage: structural protein account for 5～40%, and phosphatide accounts for 60～95%; In the red corpuscle of rebuilding, cell membrane component accounts for 5～30%, and oxyphorase accounts for 70～95%.
The HRBC of above-mentioned reconstruction in vitro is observed under greater than 100 times Electronic Speculum has the concave-concave disc form similar to the natural human red corpuscle.
For solve the problems of the technologies described above two, the technical scheme that the present invention takes is: isolating to structural protein obtain the corresponding structure protein ingredient through the protein purification system; Oxyphorase, the purified renaturation postlyophilization of structural protein is standby; Standby behind the phosphatide employing purification techniques purifying.Use the collaborative oxyphorase, external preparation artificial red blood cells sealed of membrane phospholipid-structural protein.Its technical process as shown in Figure 1, concrete preparation method's step is as follows:
1, the raw product that will come from the membranin of animal erythrocyte obtains the pure product of structural protein through separation and purification, freezing, vacuum-drying;
2, in vessel, add phosphatide in proportion, and add dissolution with solvents, pass through rotary evaporation again, remove and desolvate, obtain the immobilized artificial membrane of reconstruct;
3, in proportion with the oxyphorase physiological saline solution, and add the dissolved structural protein in proportion, slowly add in the immobilized artificial membrane of reconstruct, and seal oxyphorase by rotatable reactor;
4, the product that step 3 is obtained is suspended in the physiological saline that contains the red corpuscle nutrient solution, hatches cultivation after at least 30 minutes, has both made the artificial red blood cells of reconstruction.
For solve the problems of the technologies described above three, the technical solution used in the present invention is: in the blood substitute with the preparation of the HRBC of above-mentioned reconstruction in vitro, per-cent by volume, rebuild red corpuscle and account for 10%～55%, cell nutrient solution accounts for 45%～905%, and wherein the red corpuscle nutritive medium comprises physiological saline and nutritious protein liquid etc.
1. the present invention proposes the notion of red corpuscle reconstruction in vitro (reconstruction oferythrocyte in vitro) first; At present medically used blood substitute comprise liposomal encapsulated oxyphorase all can only be on partial function and structure normoerythrocyte in the analogue body.
2. the notion of the red corpuscle reconstruction in vitro of the present invention's proposition is based on and not only fully realizes erythrocytic biochemical functions, but also fully realizes erythrocytic biomechanics and rheologic behavio(u)r; Take into full account in design and have inseparable inner link between this cyto-mechanics characteristic and its biochemical functions.
3. the red corpuscle of reconstruction in vitro of the present invention not only will possess normal biochemical functions, and will possess the concave-concave disc form that good sex change and normoerythrocyte have.
4. this research makes by ectogenic film fat, structural protein, oxyphorase and makes up red corpuscle; The red corpuscle that makes up not only also has bigger improvement than existing blood substitute near normoerythrocyte but also its life-span on function.
Description of drawings
1, Fig. 1. be reconstruction in vitro artificial red blood cells's process flow sheet; Its technological process and method are referring to relevant embodiment.
2, Fig. 2. be the ultra-violet absorption spectrum of structural protein, comprise the ultra-violet absorption spectrum of erythrocyte membrane protein, soybean 11S sphaeroprotein.
3, Fig. 3. be structural protein-erythrocyte membrane protein gel electrophoresis UV scanning collection of illustrative plates.
4, Fig. 4. measuring the viscoelastic system diagram of artificial red blood cells for micropipette aspiration, is to write down erythrocytic deformation process under certain negative pressure by online in real time, estimates the classical experimental technique of cyto-mechanics characteristic.Experimental result demonstrates the erythrocytic deformability of reconstruction and natural normoerythrocyte does not have notable difference.
5, Fig. 5. for rebuilding artificial red blood cells's morphologic observation, rebuild the artificial red blood cells by microscopic examination and have normocytic concave-concave disc form.
6, Fig. 6. be to rebuild Oxyhemoglobins and Reduced Hbs abosrption spectrogram among the artificial red blood cells, illustrate that oxyphorase among the reconstruction artificial red blood cells has to take oxygen and oxygen release function normally.
7, Fig. 7. for rebuilding oxygen saturation curve among the artificial red blood cells, further specify the reconstruction artificial red blood cells by blood gas analysis and have normal oxygen saturation curve as blood substitute.
Embodiment
The separation purification method I of embodiment 1 structural protein
(1), extracts structural protein
Get soybean 5 grams and soaked 8～12 hours with physiological saline 10～30ml, tissue is smashed after-filtration to pieces and is removed residue; In emulsion, add 10～200 120mmol magnesium chloride normal saline solutions, when having treated that floss generates with solution frozen centrifugation (5000rpm,～4 ℃) 15～20 minutes, abandoning supernatant, precipitation is used physiological saline solution, adds ammoniumsulphate soln then to saturated coagulation floss, again by frozen centrifugation (5000rpm,～4 ℃) 15～20 minutes, obtain 11S sphaeroprotein crude product.
(2), the separation and purification of structural protein
Above-mentioned 11S sphaeroprotein crude product through the dialysis (molecular weight cut-off＜3,000) desalination to remove ionogen, lyophilize (80 ℃ 20mT), obtain the structural protein product.The plant structure albumen that obtains by this method is sphaeroprotein, is generally α β dimer, the tetramer, six aggressiveness, passes through pH (5.4～7.2) and ionic strength (Mg in the experiment
2 +: 10～200mmol) regulation and control make and are α β dimer sphaeroprotein in the product more than 70%.
The separation and purification II of embodiment 2 structural protein
(1), extracts structural protein
With sex change and the enzymolysis of the blood that contains antithrombotics (every 30ml blood adds about 5ml heparin pH7.4 isotonic phosphate buffer liquid) in order to reduce structural protein to greatest extent, entire operation should be carried out under 0～4 ℃ of low temperature.Get above-mentioned anticoagulation 5ml, frozen centrifugation (3000rpm ,～4 ℃, 15～30 minutes) makes erythroprecipitin, separated plasma supernatant liquor and buffy coat (buffy coat).
The wait phosphatizing acid buffer of red corpuscle with the precooling pH7.4 of 3 times of volumes washed 2～5 times, and each frozen centrifugation (3000rpm ,～4 ℃, 15 minutes) is removed supernatant liquor and precipitation top layer.
In the red corpuscle of cleaning, 10～50 ratio adds 5～10mmol/L of precooling by volume, and the hypotonic Tris of pH7.4 (Tutofusin tris)-HCl damping fluid places～2～12 hours haemolysis of 4 ℃ of environment; Frozen centrifugation (9000rpm ,～4 ℃, 15～30 minutes) then, abandoning supernatant is separated with oxyphorase, makes the erythrocyte membrane protein precipitation, obtains the membranin crude product.
(2), the purifying of structural protein
The membranin crude product that obtains is with Tris-HCl damping fluid washing 2～5 times, the membranin crude product through dialysis (molecular weight cut-off＜3,000) desalination to remove ionogen, lyophilize (80 ℃ 20mT), obtain the structural protein product.
The ultra-violet absorption spectrum of embodiment 3 structural protein
The structural protein product that separation and purification is obtained is dissolved in the physiological saline with the concentration of 200～800ppm, carries out ultraviolet spectro-photometric analysis.Instrument: PE-λ 900, scanning wavelength scope: 200～400nm.Spectrogram is referring to Fig. 2.The solid line representative comes from the ultraviolet absorption peak of the membranin of animal erythrocyte among the figure, and dotted line is all represented the ultraviolet absorption peak of the 11S sphaeroprotein that comes from vegetable-protein.Article three, the curve of spectrum all is presented at the 280nm place and has maximum absorption band.
The gel electrophoresis UV scanning collection of illustrative plates of embodiment 4 structural protein detects
The structural protein product that separation and purification is obtained is dissolved in the physiological saline with the concentration of 10～200g/ml, carries out the polyacrylamide gel electrophoresis analysis.Instrument: electrophoresis apparatus BIO-RAD 300; Picture system: IMAGE SYSTEM SX-100 scans gel.
Referring to Fig. 3, high-visible three protein protomer absorption peaks in gel electrophoresis UV scanning collection of illustrative plates, wherein to account for the per-cent of total protein peak area be P to the peak area at each peak
1: P 15%～26%,
2: 12%～24% for rebuilding erythrocytic main membrane skeleton protein; P
3: 15%～20% for rebuilding erythrocytic film functional protein.
Embodiment 5 makes up the proportioning and the analysis of artificial red blood cells's membrane phospholipid
The phosphatide that the present invention makes up artificial red blood cells's film is the concentrated refined plant phosphatide that commercialization is produced.
The main component per-cent of phosphatide among the embodiment:
1. phosphatidylcholine: 20～60%
2. phosphatidylethanolamine: 15～40%
3. phosphatidylinositols: 5～20%
4. phosphatidyl silk amino acid: 12～30%
Embodiment 6 rebuilds artificial red blood cells's method I
1, the structure of cytolemma
A, the refining concentrating soya lecithin of 200～500mg is placed the 50ml round-bottomed flask, with 5ml lipid lysate (chloroform/methanol: 3/1) slowly dissolving, round-bottomed flask is placed water-bath on the rotatory evaporator, open heating unit and make bath temperature reach 36 ℃, treat that starting rotatory evaporator (10rpm) after the phosphatide sample dissolution slowly is coated with phosphatide flask bottle wall at the bottom of the garden; To be coated evenly after, the temperature of water-bath is risen to 60～80 ℃, and charges into nitrogen protection, it is clean to solvent evaporates to continue rotary evaporation then, is cooled to room temperature.
B, the erythrocyte membrane protein 10～50mg of preparation is dissolved in 200 μ l etc. oozes physiological saline or isotonic phosphate buffer liquid (pH7.4), and add 10 μ l 10mmol/L oxidation inhibitor or protective material solution.After treating sample dissolution, slowly be added drop-wise at the bottom of the garden on the rotatory evaporator in the flask.
2, seal oxyphorase
With oxyphorase 20～50mg, under nitrogen protection, be dissolved in 400ml etc. and ooze physiological saline or isotonic phosphate buffer liquid (pH7.4), and add 10 μ l 10mmol/L oxidation inhibitor or protective material solution; After treating sample dissolution, slowly be added drop-wise at the bottom of the garden that has been coated with phosphatide and structural protein on the rotatory evaporator in the flask;
Under 36 ℃ of water-baths and nitrogen N 2 protections, continued rotary flask 30 minutes.After question response finishes, be transferred to reaction solution in the test tube and charge into the N2 capping.
3, hatch cultivation
Cell sample is hatched cultivation 3～30 minutes, add the red corpuscle nutrient solution of an amount of volume then and ooze the physiological saline dilution, regulate and rebuild erythrocytic hematocrit 35～45%; It is standby to be stored in 4 ℃ of refrigerator cold-storages.
Embodiment 7 rebuilds artificial red blood cells's method II
(1), the structure of cytolemma
A, 200～500mg phosphatidylcholine and 10mg cholesterol are placed the 50ml round-bottomed flask, with 5ml lipid lysate (chloroform/methanol: 3/1) slowly dissolving, round-bottomed flask is placed water-bath on the rotatory evaporator, open heating unit and make bath temperature reach 36 ℃, treat that starting rotatory evaporator (10rpm) after the phosphatide sample dissolution slowly is coated with phosphatide flask bottle wall at the bottom of the garden; To be coated evenly after, the temperature of water-bath is risen to 60～80 ℃, and charges into nitrogen protection, it is clean to solvent evaporates to continue rotary evaporation then, is cooled to room temperature.
B, the plant 11S sphaeroprotein 10～50mg of preparation is dissolved in 200 μ l etc. oozes physiological saline or isotonic phosphate buffer liquid (pH7.4), and add 10 μ l 10mmol/L oxidation inhibitor or protective material solution.After treating sample dissolution, slowly be added drop-wise at the bottom of the garden on the rotatory evaporator in the flask.
(2), seal oxyphorase
With oxyphorase 20～50mg, under nitrogen protection, be dissolved in 400ml etc. and ooze physiological saline or isotonic phosphate buffer liquid (pH7.4), and add 10 μ l 10mmol/L oxidation inhibitor or protective material solution; After treating sample dissolution, slowly be added drop-wise at the bottom of the garden that has been coated with phosphatide and structural protein on the rotatory evaporator in the flask; Under 36 ℃ of water-baths and nitrogen N 2 protections, continued rotary flask 30 minutes.After question response finishes, be transferred to reaction solution in the test tube and charge into the N2 capping.
(3), hatch cultivation
Cell sample hatched cultivated 3～30 minutes, add the red corpuscle nutrient solution of an amount of volume then and wait and ooze the physiological saline dilution, regulate the erythrocytic hematocrit of reconstruction 35～45%; It is standby to be stored in 4 ℃ of refrigerator cold-storages.
Embodiment 8 rebuilds the artificial red blood cells and has normocytic deformability experiment
Artificial red blood cells's deformability is described with erythrocytic film visco-elasticity, adopts micropipette aspiration system (referring to Fig. 4) to measure artificial red blood cells's visco-elasticity.The artificial red blood cells suspends with physiological saline and places Xiao Chi, and fills with whole microtubule system with physiological saline; Move the surface of microtubule with micromanipulation system, produce a step negative pressure by pressure system and suck the artificial red blood cells to the artificial red blood cells that suspends; Make the small portion of cell be inhaled into microtubule, with shooting processing instrument, picture recording system and markers record artificial red blood cells deformation time process with negative pressure; The length and the each point that are inhaled into microtubule by replay image treatment system mensuration microtubule radius R p, artificial red blood cells get time course.Adopt Kelvin model and Chien " calotte submodel " to describe artificial red blood cells's visco-elasticity.
Purchase theory according to viscoelastic of erythrocyte membrane, with kelvin model-fitting experimental result.The viscosity unit that this model is η by the modulus Flexible element that is μ and coefficient of viscosity is formed in parallel.
Be located at t constantly, the length that red corpuscle is inhaled into microtubule is Dp, at t
1In writing time, the maximum value that it reached is Dpm, and following relation is then arranged.
(ΔP)R
p/μ＝2X
m-1+ln(2X
m)
τ＝-a/|4[d?ln(D
pm-D
p)/dt]
η＝μt
Wherein: Δ P is the step negative pressure, and Rp is the microtubule radius, and Xm is Dpm/Dp, т by the time constant of record deformation process, a is a fitting constant.
(ΔP)R
P/μ＝2X-1+ln(2X)
＝aX+b
Data and above-mentioned formula can calculate the Young's modulus and the coefficient of viscosity (referring to table 1) of trying to achieve the artificial red blood cells by experiment, as the physical quantity that characterizes artificial red blood cells's deformability.
Table 1. artificial red blood cells viscoelastic coefficient
Young's modulus μ coefficient of viscosity η
The red corpuscle type
(10
-3dyn/cm) (10
-4dyn·s/cm)
Rebuild artificial red blood cells 4.562～7.892 0.175～0.372
Natural normoerythrocyte 1.120～8.753 0.185～0.452
Table 1 data presentation goes out to rebuild erythrocytic deformability and natural normoerythrocyte does not have notable difference.
Embodiment 9 rebuilds the observation that the artificial red blood cells has normocytic concave-concave disc form
The artificial red blood cells constructed according to foregoing method should have the concave-concave disc form (referring to Fig. 5) that normoerythrocyte has.
Rebuild artificial red blood cells (Fig. 5 A, Fig. 5 C), its form almost with the plesiomorphism of normoerythrocyte (Fig. 5 D), all have concave-concave disc form; The cell yardstick near or less than normoerythrocyte.Fig. 5 B is micropipette aspiration systems measurement artificial red blood cells's a viscoelastic experiment picture.
What embodiment 10 rebuild artificial red blood cellses takes oxygen-oxygen release function
According to the constructed artificial red blood cells of foregoing method, have and take oxygen-oxygen release function (referring to Fig. 6,7) normally.Fig. 6 shows the difference of oxyphorase absorption spectrum under oxygen saturation and anoxia condition among the artificial red blood cells of reconstruction, reflects that the oxyphorase of sealing has to take oxygen-oxygen release function normally.When Fig. 7 shows usefulness reconstruction artificial red blood cells as blood substitute, under different oxygen partial pressure conditions, the function of carrying oxygen of artificial blood.
Embodiment 11 rebuilds the experimentation on animals application example 1 of artificial red blood cells as blood substitute
To rebuild the artificial red blood cells is raw material, is equipped with physiological saline and adds 10～20% calf serum to be re-dubbed artificial blood as blood substitute; The action thing of going forward side by side is tested.
Rebuild the artificial red blood cells: 10～55% (volume percent).
The red corpuscle nutritive medium: 45～90% (volume percent), wherein contain the calf serum of 10～20% (volume percent).
Laboratory animal: male Wister rat, 5 every group, body weight is in 185～250 gram scopes.
Experimentation: with the consumption of 1% Veronal sodium solution with body weight 5 ‰, the abdominal cavity injects the Wister rat, standard law sterilization, preserved skin, jugular vein blood sampling 1ml; Experimental group is injected 1ml with rebuilding the blood substitute that the artificial red blood cells prepares then; Control group injects the physiological saline that 1ml contains calf serum.The ligation blood vessel is sewed up a wound; Inject the gentamicin preventing infection, quarantine; Surviving rate: experimental group 100%, control group 80% (1 rat hemorrhagic shock death).The maximum rate of displacement of blood is Q volume of blood～10%, and the rat blood circulation of displacement back, blood oxygen saturation, breathing and physiological activity are all normal.
Embodiment 12 rebuilds the experimentation on animals application example 2 of artificial red blood cells as blood substitute
To rebuild the artificial red blood cells is raw material, is equipped with physiological saline and adds 10～20% calf serum to be re-dubbed artificial blood as blood substitute; The action thing of going forward side by side is tested.
Rebuild the artificial red blood cells: 10～55% (volume percent).
The red corpuscle nutritive medium: 45～90% (volume percent), wherein contain the calf serum of 10～20% (volume percent).
Laboratory animal: male Wister rat, 5 every group, body weight is in 185～250 gram scopes.
Experimentation: with the consumption of 1% Veronal sodium solution with body weight 5 ‰, the abdominal cavity injects the Wister rat, standard law sterilization, preserved skin, jugular vein blood sampling 2ml; Experimental group is injected 2ml with rebuilding the blood substitute that the artificial red blood cells prepares then; Control group injects the physiological saline that 2ml contains calf serum.The ligation blood vessel is sewed up a wound; Inject the gentamicin preventing infection, quarantine; Surviving rate: experimental group 100%, control group 40% (1 rat hemorrhagic shock death, respiratory distress syndrome (ivrds) appears in 2 rats, and is last dead).The maximum rate of displacement of blood is Q volume of blood～20%, and the rat blood circulation of displacement back, blood oxygen saturation, breathing and physiological activity are all normal.
Embodiment 13 rebuilds the experimentation on animals application example 3 of artificial red blood cells as blood substitute
To rebuild the artificial red blood cells is raw material, is equipped with physiological saline and adds 10～20% calf serum to be re-dubbed artificial blood as blood substitute; The action thing of going forward side by side is tested.
Rebuild the artificial red blood cells: 10～55% (volume percent).
The red corpuscle nutritive medium: 45～90% (volume percent), wherein contain the calf serum of 10～20% (volume percent).
Laboratory animal: male Wister rat, 5 every group, body weight is in 185～250 gram scopes.
Experimentation: with the consumption of 1% Veronal sodium solution with body weight 5 ‰, the abdominal cavity injects the Wister rat, standard law sterilization, preserved skin, jugular vein blood sampling 3ml; Experimental group is injected 3ml with rebuilding the blood substitute that the artificial red blood cells prepares then; Control group injects the physiological saline that 3ml contains calf serum.The ligation blood vessel is sewed up a wound; Inject the gentamicin preventing infection, quarantine; Surviving rate: experimental group 100%, control group 20% (1 rat hemorrhagic shock death, respiratory distress syndrome (ivrds) appears in 3 rats, and is last dead).The maximum rate of displacement of blood is Q volume of blood～30%, and the rat blood circulation of displacement back, breathing and physiological activity are all normal.
Embodiment 14 rebuilds the experimentation on animals application example 4 of artificial red blood cells as blood substitute
To rebuild the artificial red blood cells is raw material, is equipped with physiological saline and adds 10～20% calf serum to be re-dubbed artificial blood as blood substitute; The action thing of going forward side by side is tested.
Rebuild the artificial red blood cells: 10～55% (volume percent).
The red corpuscle nutritive medium: 45～90% (volume percent), wherein contain the calf serum of 10～20% (volume percent).
Laboratory animal: male Wister rat, 5 every group, body weight is in 185～250 gram scopes.
Experimentation: with the consumption of 1% Veronal sodium solution with body weight 5 ‰, the abdominal cavity injects the Wister rat, standard law sterilization, preserved skin, jugular vein blood sampling 4ml; Experimental group is injected 4ml with rebuilding the blood substitute that the artificial red blood cells prepares then; Control group injects the physiological saline that 4ml contains calf serum.The ligation blood vessel is sewed up a wound; Inject the gentamicin preventing infection, quarantine; Surviving rate: experimental group 100%, control group 0% (5 rats hemorrhagic shock all occurs and respiratory distress syndrome (ivrds) occurs, and are last dead).The maximum rate of displacement of blood is Q volume of blood～40%, and the rat blood circulation of displacement back, breathing and physiological activity are all normal.
Embodiment 15 rebuilds the refrigeration experiment behind the composite blood substitute of artificial red blood cells
Be equipped with physiological saline and stablizer and oxidation inhibitor and add 10～20% calf serum and be re-dubbed artificial blood rebuilding the artificial red blood cells as blood substitute; Place 4 ℃ and-20 ℃ of refrigerators to carry out substep respectively and refrigerate experiment in 4～72 hours.Thaw after the recovery, rebuild the artificial red blood cells and still have normal concave-concave disc form; And the artificial red blood cells after the experimentation on animals explanation refrigeration so can be used as the effective blood surrogate.
Laboratory animal: male Wister rat, 5 every group, body weight is in 185～250 gram scopes.
Experimentation: with the consumption of 1% Veronal sodium solution with body weight 5 ‰, the abdominal cavity injects the Wister rat, standard law sterilization, preserved skin, jugular vein blood sampling 1～3ml; Experimental group is injected 3ml with rebuilding the blood substitute that the artificial red blood cells prepares then; Control group injects the physiological saline that 3ml contains calf serum.The ligation blood vessel is sewed up a wound; Inject the gentamicin preventing infection, quarantine; Surviving rate: experimental group 100%.The maximum rate of displacement of blood is 10～30% of a Q volume of blood, and the rat blood circulation of displacement back, breathing and physiological activity are all normal.
Claims (4)
1, a kind of HRBC of reconstruction in vitro is characterized in that: comprise following material:
(1). oxyphorase;
(2). structural protein: for coming from the membranin of people, ox, sheep, pig erythrocyte; Or come from natural plant protein product such as glycinin 11S; Its molecular weight: 250,000～620,000 dalton; Its ultra-violet absorption spectrum is: λ
Max=280nm (physiological saline); High-visible three protein protomer absorption peaks in gel electrophoresis UV scanning collection of illustrative plates, wherein to account for the per-cent of total protein peak area be 15%～26%, 12%～24% and 15%～20% to the peak area at each peak;
(3). phosphatide: come from the natural phant concentrated phosphatide, obtain product through separation and purification; Or the phosphatide product that obtains by microbial fermentation technology;
In the mentioned component, structural protein and phosphatide constitute erythrocytic cell membrane component, wherein structural protein 5%～40%, phosphatide: 60%～95%; Rebuild in the red corpuscle cell membrane component 5%～30%, oxyphorase 70%～95%;
The artificial red blood cells of above-mentioned reconstruction in vitro observes at inverted microscope (100 times) has the concave-concave disc form similar to the natural human red corpuscle.
2, a kind of artificial red blood cells's according to claim 1 of reconstruction in vitro method, its method steps is as follows:
(1). will come from the membranin of animal erythrocyte or come from the natural plant protein product such as the raw product of glycinin 11S obtains the pure product of structural protein through separation and purification, freezing, vacuum-drying;
(2). add phosphatide in proportion, and add dissolution with solvents, pass through rotary evaporation again, remove and desolvate, obtain the immobilized artificial membrane of reconstruct;
(3). in proportion with the oxyphorase physiological saline solution, and add the dissolved structural protein in proportion, slowly add in the immobilized artificial membrane of reconstruct, and seal oxyphorase by rotatable reactor;
(4). the product that step 3 is obtained is suspended in the physiological saline that contains the red corpuscle nutrient solution, hatches cultivation after at least 30 minutes, has both made the artificial red blood cells of reconstruction.
3, the blood substitute of a kind of HRBC of reconstruction in vitro as claimed in claim 1 preparation is characterized in that: per-cent by volume, and rebuild red corpuscle and account for 10%～55%, red corpuscle nutritive medium 45%～90%.
4, the artificial red blood cells of reconstruction in vitro according to claim 1, it is characterized in that: the main component weight percent of described phosphatide: phosphatidylcholine: 20～60%, phosphatidylethanolamine: 15～40%, phosphatidylinositols: 5～20%, phosphatidyl silk amino acid: 12～30%.
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